Dirofilaria and brugia thioredoxin peroxidase type-2 (TPX-2) nucleic acid molecules, and uses thereof

ABSTRACT

The present invention relates to: Dirofilaria and Brugia thioredoxin peroxidase type-2 (TPx-2) proteins; Dirofilaria and Brugia TPx-2 nucleic acid molecules, including those that encode such TPx-2 proteins; antibodies raised against such TPx-2 proteins; and compounds that inhibit Dirofilaria and Brugia TPx-2 activity. The present invention also includes methods to obtain such proteins, nucleic acid molecules, antibodies, and inhibitory compounds. Also included in the present invention are therapeutic compositions comprising such proteins, nucleic acid molecules, antibodies and/or inhibitory compounds as well as the use of such therapeutic compositions to protect animals from diseases caused by parasitic helminths.

FIELD OF THE INVENTION

The present invention relates to Dirofilaria and Brugia thioredoxinperoxidase (TPx-2) nucleic acid molecules, proteins encoded by suchnucleic acid molecules, antibodies raised against such proteins, andinhibitors of such proteins. The present invention also includestherapeutic compositions comprising such nucleic acid molecules,proteins, antibodies, and/or inhibitors, as well as their use to protectanimals from diseases caused by parasitic helminths, such as heartwormdisease, elephantiasis, and hydrocele.

BACKGROUND OF THE INVENTION

Parasitic helminth infections in animals, including humans, aretypically treated by chemical drugs. One disadvantage with chemicaldrugs is that they must be administered often. For example, dogssusceptible to heartworm are typically treated monthly. Repeatedadministration of drugs, however, often leads to the development ofresistant helminth strains that no longer respond to treatment.Furthermore, many of the chemical drugs cause harmful side effects inthe animals being treated, and as larger doses become required due tothe build up of resistance, the side effects become even greater.Moreover, a number of drugs only treat symptoms of a parasitic diseasebut are unable to prevent infection by the parasitic helminth.

An alternative method to prevent parasitic helminth infection includesadministering a vaccine against a parasitic helminth. Although manyinvestigators have tried to develop vaccines based on specific antigens,it is well understood that the ability of an antigen to stimulateantibody production does not necessarily correlate with the ability ofthe antigen to stimulate an immune response capable of protecting ananimal from infection, particularly in the case of parasitic helminths.Although a number of prominent antigens have been identified in severalparasitic helminths, including in Dirofilaria and Brugia, there is yetto be a commercially available vaccine developed for any parasitichelminth.

As an example of the complexity of parasitic helminths, the life cycleof D. immitis, the helminth that causes heartworm disease, includes avariety of life forms, each of which presents different targets, andchallenges, for immunization. In a mosquito, D. immitis microfilariae gothrough two larval stages (L1 and L2) and become mature third stagelarvae (L3), which can then be transmitted back to the dog when themosquito takes a blood meal. In a dog, the L3 molt to the fourth larvalstage (L4), and subsequently to the fifth stage, or immature adults. Theimmature adults migrate to the heart and pulmonary arteries, where theymature to adult heartworms. Adult heartworms are quite large andpreferentially inhabit the heart and pulmonary arteries of an animal.Sexually mature adults, after mating, produce microfilariae whichtraverse capillary beds and circulate in the vascular system of the dog.

In particular, heartworm disease is a major problem in dogs, whichtypically do not develop immunity, even upon infection (i.e., dogs canbecome reinfected even after being cured by chemotherapy). In addition,heartworm disease is becoming increasingly widespread in other companionanimals, such as cats and ferrets. D. immitis has also been reported toinfect humans.

As such, there remains a need to identify an efficacious compositionthat protects animals against diseases caused by parasitic helminths,such as heartworm disease. Preferably, such a composition also protectsanimals from infection by such helminths.

Prior studies have shown that larval stages of parasitic helminths aresusceptible to antibody dependent cellular cytotoxicity (ADCC) in vitro.ADCC reactions mainly involve phagocytes such as macrophages,eosinophils and neutrophils. These cells are known to generate reactiveoxygen species, such as hydroperoxides and free radicals, which candamage parasites. As a defense, parasites have evolved a number ofantioxidant enzymes to overcome the damaging effects of reactive oxygenspecies generated by the host. While not being bound by theory, suchparasitic helminth antioxidant enzymes are attractive targets forvaccines and other chemotherapeutic agents useful in the prevention ortreatment of parasitic diseases.

Thioredoxin peroxidases (TPx, previously called thiol-specificantioxidants, or TSA) are newly discovered antioxidant enzymes.Antioxidants are involved in detoxification of reactive oxygen andsulfur species. Recent studies indicate that TPx proteins are involvedin reducing hydroperoxides and lipid peroxides with thioredoxin as anintermediate donor. Prior investigators have identified yeast TPxproteins; and have cloned several mammalian TPx genes and a protozoanTPx gene. See, for example, Yamamoto et al, 1989, Gene 80, 337-343,Torian et al., 1990, Proc. Natl. Acad. Sci. USA 87, 6358-6362, Reed etal., 1992, Infection and Immunity. 60, 542-549, Ramussen et al, 1992,Electrophoresis 13, 960-969, Tannich et al., 1993, Trop. Med. Parasitol.44, 116-118, Prosperi et al., 1993, J. Biol. Chem. 268, 11050-11056,Ishii et al., 1993, J. Biol. Chem. 268, 18633-18636, Chae et al., 1993,J. Biol. Chem. 268, 16815-16821, Ishii et al., 1993, J. Biol. Chem. 268,18633-18636, Chae et al., 1994, Proc. Natl. Acad. Sci. USA 91,7022-7026, Kawai et al, 1994, J. Biochem. 115, 641-643, Chae et al,1994, Proc. Natl. Acad. Sci. USA 91, 7017-7021 and Chae et al, 1994,Biofactors 4, 177-180. In addition, the nucleic acid and deduced aminoacid sequences of an adult Onchocerca volvulus TPx have been determined;see GenBank™ Accession No. U31052, and Chandrashekar, et al., Feb. 22,1996, Abstract 203, "Molecular Helminthology: An Integrated Approach",Keystone Symposia. A distantly-related larval thioredoxin peroxidasenucleic acid molecule (TPx-1) was recently isolated from D. immitis; seeU.S. patent application Ser. No. 08/602,010, by Tripp, et al., filedFeb. 15, 1996, and Tripp, et al., Feb. 22, 1996, Abstract 214,"Molecular Helminthology: An Integrated Approach", Keystone Symposia.Patent application Ser. No. 08/602,010, ibid., is incorporated byreference herein in its entirety. Although yeast, human and bovinecortex TPx proteins have been shown to have thioredoxin peroxidaseactivity (see, for example, Sauri et al, 1995, Biochem. Biophys. Res.Comm. 208, 964-969; and Watabe et al, 1995, Biochem. Biophys. Res. Comm.213, 1010-1016), the other TPx genes or proteins have been designated assuch only by nucleic acid sequence homology or by the binding ofspecific antibodies.

SUMMARY OF THE INVENTION

The present invention relates to a novel product and process to protectanimals against parasitic helminth infection (e.g., to prevent and/ortreat such an infection). The present invention provides Dirofilaria andBrugia thioredoxin peroxidase type-2 (TPx-2) proteins and mimetopesthereof; Dirofilaria and Brugia TPx-2 nucleic acid molecules, includingthose that encode such proteins; antibodies raised against such TPx-2proteins (anti-Dirofilaria and anti-Brugia TPx-2 antibodies); andcompounds that inhibit TPx-2 activity (i.e, inhibitory compounds orinhibitors).

The present invention also includes methods to obtain such proteins,nucleic acid molecules, antibodies and inhibitory compounds. Alsoincluded in the present invention are therapeutic compositionscomprising such proteins, nucleic acid molecules, antibodies, andinhibitory compounds, as well as use of such therapeutic compositions toprotect animals from diseases caused by parasitic helminths.

One embodiment of the present invention is an isolated nucleic acidmolecule which includes a Dirofilaria TPx-2 nucleic acid molecule or aBrugia TPx-2 nucleic acid molecule. Such nucleic acid molecules arereferred to as TPx-2 nucleic acid molecules. A preferred isolatednucleic acid molecule of this embodiment includes a Dirofilaria immitis(D. immitis) TPx-2 nucleic acid molecule or a Brugia malayi (B. malayi)TPx-2 nucleic acid molecule. A D. immitis TPx-2 nucleic acid moleculepreferably includes nucleic acid sequence SEQ ID NO:1, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:20, or SEQ IDNO:21, and a B. malayi TPx-2 nucleic acid molecule preferably includesnucleic acid sequence SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:11 or SEQ IDNO:12.

The present invention also relates to recombinant molecules, recombinantviruses and recombinant cells that include an isolated TPx-2 nucleicacid molecule of the present invention. Also included are methods toproduce such nucleic acid molecules, recombinant molecules, recombinantviruses and recombinant cells.

Another embodiment of the present invention includes a Dirofilaria TPx-2protein or a Brugia TPx-2 protein. A preferred TPx-2 protein includes aD. immitis TPx-2 protein or a B. malayi TPx-2 protein. A preferred D.immitis TPx-2 protein comprises amino acid sequence SEQ ID NO:2, and apreferred B. malayi TPx-2 protein comprises amino acid sequence SEQ IDNO:9.

The present invention also relates to: mimetopes of either Dirofilariaor Brugia TPx-2 proteins; isolated antibodies that selectively bind toDirofilaria or Brugia TPx-2 proteins or mimetopes thereof; andinhibitors of Dirofilaria or Brugia TPx-2 proteins or mimetopes thereof.Also included are methods, including recombinant methods, to produceproteins, mimetopes, antibodies, and inhibitors of the presentinvention.

Another embodiment of the present invention is a method to identify acompound capable of inhibiting parasitic helminth TPx-2 activity,comprising the steps of: (a) contacting a Dirofilaria or a Brugia TPx-2protein with a putative inhibitory compound under conditions in which,in the absence of the compound, the protein has TPx-2 activity; and (b)determining if the putative inhibitory compound inhibits the TPx-2activity. Also included in the present invention is a test kit toidentify a compound capable of inhibiting parasitic helminth TPx-2activity. Such a test kit includes a Dirofilaria or a Brugia TPx-2protein having TPx-2 activity and a means for determining the extent ofinhibition of the TPx-2 activity in the presence of a putativeinhibitory compound.

Yet another embodiment of the present invention is a therapeuticcomposition that is capable of protecting an animal from disease causedby a parasitic helminth. Such a therapeutic composition includes one ormore of the following protective compounds: an isolated Dirofilaria orBrugia TPx-2 protein or a mimetope thereof; an isolated Dirofilaria orBrugia TPx-2 nucleic acid molecule; an isolated antibody thatselectively binds to a Dirofilaria or a Brugia TPx-2 protein; or aninhibitor of TPx-2 protein activity identified by its ability to inhibitDirofilaria or Brugia TPx-2 activity. A preferred therapeuticcomposition of the present invention also includes an excipient, anadjuvant, or a carrier. Preferred TPx-2 nucleic acid moleculetherapeutic compositions of the present invention include geneticvaccines, recombinant virus vaccines, and recombinant cell vaccines.Also included in the present invention is a method to protect an animalfrom disease caused by a parasitic helminth, comprising the step ofadministering to the animal a therapeutic composition of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for isolated Dirofilaria and Brugiathioredoxin peroxidase type-2 (TPx-2) proteins, isolated Dirofilaria andBrugia TPx-2 nucleic acid molecules, antibodies directed againstDirofilaria and Brugia TPx-2 proteins, and other inhibitors of parasitichelminth TPx-2 activity. As used herein, the terms isolated DirofilariaTPx-2 proteins, isolated Brugia TPx-2 proteins, isolated DirofilariaTPx-2 nucleic acid molecules, and isolated Brugia TPx-2 nucleic acidmolecules refers to TPx-2 proteins and TPx-2 nucleic acid moleculesderived from parasitic helminths of the genera Dirofilaria and Brugiaand, as such, can be obtained from their natural source, or can beproduced using, for example, recombinant nucleic acid technology orchemical synthesis. Also included in the present invention is the use ofthese proteins, nucleic acid molecules, antibodies and other inhibitorsas therapeutic compositions to protect animals from parasitic helminthdiseases as well as in other applications, such as those disclosedbelow.

Dirofilaria and Brugia TPx-2 proteins and nucleic acid molecules of thepresent invention have utility because they represent novel targets foranti-parasite vaccines and drugs. The products and processes of thepresent invention are advantageous because they enable the inhibition ofparasite defense mechanisms that involve antioxidants such as TPx-2.While not being bound by theory, it is believed that TPx-2 proteins candefend parasitic helminths from reactive oxygen radical damage ofproteins, DNA, or lipids by inhibiting oxygen (O₂) radical-dependentinactivation of parasite cellular enzymes.

Dirofilaria and Brugia TPx-2 proteins and nucleic acid molecules of thepresent invention are different from recently isolated D. immitis TPx-1proteins and nucleic acid molecules (see patent application Ser. No.08/602,010, ibid.) in several ways. TPx-2 proteins and nucleic acidmolecules of the present invention have very divergent amino acid andnucleotide sequences relative to the previously disclosed TPx-1 aminoacid and nucleotide sequences. In addition, TPx-2 proteins of thepresent invention have considerably lower predicted and measuredisoelectric points (pI) than the predicted pI of the D. immitis TPx-1protein. Furthermore, the D. immitis TPx-1 protein does not appear to bereleased as an excretory-secretory (E-S) product in the larval stages,while the D. immitis TPx-2 protein, as described in the Examples, isfound in larval E-S products.

Furthermore, Dirofilaria and Brugia TPx-2 proteins and nucleic acidmolecules of the present invention can be differentiated from thepreviously disclosed adult O. volvulus TPx protein and nucleic acidmolecule (see Genbank™ Accession No. U31052 and Chandrashekar, et al.,ibid.). Chandrashekar, et al., ibid. discloses an O. volvulus TPxprotein and nucleic acid molecule isolated from adult worms, and teachesthat this TPx is the adult form of thioredoxin peroxidase, as opposed toa significantly divergent larval form of thioredoxin peroxidase isolatedfrom O. volvulus. The present invention discloses that Dirofilaria andBrugia TPx-2 proteins and nucleic acid molecules can be isolated fromlarval stages as well as from adult worms. To the inventors' knowledge,the present invention is the first disclosure of a protein or nucleicacid molecule with significant similarity to TPx-2 being isolated from alarval parasitic helminth.

One embodiment of the present invention is an isolated proteincomprising a Dirofilaria TPx-2 protein or a Brugia TPx-2 protein. It isto be noted that the term "a" or "an" entity refers to one or more ofthat entity; for example, a protein refers to one or more proteins or atleast one protein. As such, the terms "a" (or "an"), "one or more" and"at least one" can be used interchangeably herein. It is also to benoted that the terms "comprising", "including", and "having" can be usedinterchangeably. According to the present invention, an isolated, orbiologically pure, protein, is a protein that has been removed from itsnatural milieu. As such, "isolated" and "biologically pure" do notnecessarily reflect the extent to which the protein has been purified.An isolated protein of the present invention can be obtained from itsnatural source, can be produced using recombinant DNA technology or canbe produced by chemical synthesis.

As used herein, an isolated Dirofilaria or Brugia TPx-2 protein can be afull-length protein or any homolog of such a protein. An isolatedprotein of the present invention, including a homolog, can be identifiedin a straight-forward manner by the protein's ability to elicit animmune response against a parasitic helminth TPx-2 protein, to reduceperoxide, or to bind to immune serum. Examples of Dirofilaria and BrugiaTPx-2 homologs include Dirofilaria and Brugia TPx-2 proteins in whichamino acids have been deleted (e.g., a truncated version of the protein,such as a peptide), inserted, inverted, substituted and/or derivatized(e.g., by glycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitoylation, amidation, or addition ofglycerophosphatidyl inositol) such that the homolog includes at leastone epitope capable of eliciting an immune response against aDirofilaria or Brugia TPx-2 protein. That is, when the homolog isadministered to an animal as an immunogen, using techniques known tothose skilled in the art, the animal will produce an immune responseagainst at least one epitope of a natural Dirofilaria or Brugia TPx-2protein. As used herein, the term "epitope" refers to the smallestportion of a protein or other antigen capable of selectively binding tothe antigen binding site of an antibody or a T-cell receptor. It is wellaccepted by those skilled in the art that the minimal size of a proteinepitope is about four amino acids. The ability of a protein to effect animmune response can be measured using techniques known to those skilledin the art.

Dirofilaria and Brugia TPx-2 protein homologs of the present inventionalso include Dirofilaria and Brugia TPx-2 proteins that reduce peroxideand/or that bind to immune serum. Examples of methods to measure suchactivities are disclosed herein, and are known to those skilled in theart. Methods to produce and use immune serum are disclosed, for example,in Grieve et al., PCT Publication No. WO 94/15593, published Jul. 21,1994, which is incorporated by reference herein in its entirety.

Dirofilaria and Brugia TPx-2 protein homologs can be the result ofnatural allelic variation or natural mutation. Dirofilaria and BrugiaTPx-2 protein homologs of the present invention can also be producedusing techniques known in the art including, but not limited to, directmodifications to the protein or modifications to the gene encoding theprotein using, for example, classic or recombinant DNA techniques toeffect random or targeted mutagenesis.

A TPx-2 protein of the present invention is encoded by a DirofilariaTPx-2 nucleic acid molecule or a Brugia TPx-2 nucleic acid molecule. Asused herein, a Dirofilaria or Brugia TPx-2 nucleic acid moleculeincludes a nucleic acid sequence related to a natural Dirofilaria orBrugia TPx-2 gene, and preferably, to a D. immitis or to a B. malayiTPx-2 gene. As used herein, a Dirofilaria or Brugia TPx-2 gene includesall regions that control production of the Dirofilaria or Brugia TPx-2protein encoded by the gene (such as, but not limited to, transcription,translation or post-translation control regions) as well as the codingregion itself, and any introns or non-translated coding regions. As usedherein, a gene that "includes" or "comprises" a nucleic acid sequencemay include that sequence in one contiguous array, or may include thatsequence as fragmented exons. As used herein, the term "coding region"refers to a continuous linear array of nucleotides that translates intoa protein. A full-length coding region is that coding region which istranslated into a full-length, i.e., a complete, protein as would beinitially translated in its natural milieu, prior to anypost-translational modifications.

In one embodiment, a D. immitis TPx-2 gene of the present inventionincludes the nucleic acid sequence SEQ ID NO:1, as well as thecomplement of SEQ ID NO:1. Nucleic acid sequence SEQ ID NO:1 representsthe deduced sequence of the coding strand of the apparent coding regionof a CDNA (complementary DNA) nucleic acid molecule denoted herein asnDiTPx2₈₀₂, the production of which is disclosed in the Examples. Thecomplement of SEQ ID NO:1 (represented herein by SEQ ID NO:3) refers tothe nucleic acid sequence of the strand complementary to the strandhaving SEQ ID NO:1, which can easily be determined by those skilled inthe art. Likewise, a nucleic acid sequence complement of any nucleicacid sequence of the present invention refers to the nucleic acidsequence of the nucleic acid strand that is complementary to (i.e., canform a double helix with) the strand for which the sequence is cited.

In another embodiment, a B. malayi TPx-2 gene of the present inventionincludes the nucleic acid sequence SEQ ID NO:8, as well as thecomplement of SEQ ID NO:8 (represented herein by SEQ ID NO:10). Nucleicacid sequence SEQ ID NO:8 represents the deduced sequence of the codingstrand of the apparent coding region of a cDNA (complementary DNA)nucleic acid molecule denoted herein as nBmTPx2₇₃₆, the production ofwhich is disclosed in the Examples.

In another embodiment, a D. immitis TPx-2 gene or a B. malayi TPx-2 genecan be an allelic variant that includes a similar, but not identical,sequence to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:10, or anyother nucleic acid sequence cited herein. For example, an allelicvariant of a D. immitis TPx-2 gene including SEQ ID NO: 1 and SEQ IDNO:3 is a gene that occurs at essentially the same locus (or loci) inthe genome as the gene including SEQ ID NO:1 and SEQ ID NO:3, but which,due to natural variations caused by, for example, mutation orrecombination, has a similar but not identical sequence. Because naturalselection typically selects against alterations that affect function, anallelic variant usually encodes a protein having a similar activity orfunction to that of the protein encoded by the gene to which it is beingcompared. An allelic variant of a gene or nucleic acid molecule can alsocomprise alterations in the 5' or 3' untranslated regions of the gene(e.g., in regulatory control regions), or can involve alternativesplicing of a nascent transcript, thereby bringing alternative exonsinto juxtaposition. Allelic variants are well known to those skilled inthe art and would be expected to be found within a given parasitichelminth such as Dirofilaria or Brugia, since the respective genomes arediploid, and sexual reproduction will result in the reassortment ofalleles.

The minimal size of a TPx-2 protein homolog of the present invention isa size sufficient to be encoded by a nucleic acid molecule capable offorming a stable hybrid (i.e., hybridize under stringent hybridizationconditions) with the complementary sequence of a nucleic acid moleculeencoding the corresponding natural protein. As used herein, "stringenthybridization conditions" refer to those experimental conditions underwhich nucleic acid molecules having similar nucleic acid sequences willanneal to each other. Stringent hybridization conditions, as definedherein, permit the hybridization of nucleic acid molecules having atleast about 70% nucleic acid sequence identity with the nucleic acidmolecule being used as a probe in the hybridization reaction, i.e.,permit the hybridization of a nucleic acid molecule to a probe having upto about 30% base-pair mismatch. Formulae to calculate the appropriatehybridization and wash conditions to achieve hybridization permitting30% or less mismatch between two nucleic acid molecules are disclosed,for example, in Meinkoth et al, 1984, Anal. Biochem 138, 267-284;Meinkoth et al, ibid, is incorporated by reference herein in itsentirety. The size of a nucleic acid molecule encoding such a proteinhomolog is dependent on the nucleic acid composition and the percenthomology between the nucleic acid molecule and complementary sequence.It should also be noted that the extent of homology required to form astable hybrid can vary depending on whether the homologous sequences areinterspersed throughout a given nucleic acid molecule or are clustered(i.e., localized) in distinct regions on a given nucleic acid molecule.The minimal size of such a nucleic acid molecule is typically at leastabout 12 to about 15 nucleotides in length if the nucleic acid moleculeis GC-rich and at least about 15 to about 17 bases in length if it isAT-rich. As such, the minimal size of a nucleic acid molecule used toencode a TPx-2 protein homolog of the present invention is from about 12to about 18 nucleotides in length. Thus, the minimal size of a TPx-2protein homolog of the present invention is from about 4 to about 6amino acids in length. There is no limit, other than a practical limit,on the maximal size of such a nucleic acid molecule in that the nucleicacid molecule can include a portion of a gene, an entire gene, ormultiple genes. The preferred size of a protein encoded by a nucleicacid molecule of the present invention depends on whether a full-length,fusion, multivalent, or functional portion of such a protein is desired.

One embodiment of the present invention includes a Dirofilaria or BrugiaTPx-2 protein having TPx-2 enzyme activity. Such a TPx-2 proteinpreferably includes the conserved N-terminal cysteine (Cys) residuecorresponding to the Cys at position 47 in the yeast TPx protein. TheN-terminal Cys residue in the yeast TPx has been shown to be involved insubstrate peroxide reduction. Inhibition of TPx activity byN-ethylmaleimide (NEM), a compound that binds strongly to reducedcysteine residues, further indicates that the N-terminal Cys residue isa major component of the active site. Methods to detect thioredoxinperoxidase activity are described in the Examples section, as well as,for example, in Rhee et al., 1994, Mol. Cells 4: 137-142; Lim et al,1993, Biochem. Biophys. Res. Comm. 192, 273-280; Sauri et al, ibid.; andKim et al, 1988, J. Biol. Chem. 263, 4704-4711. Rhee, et al., ibid.,Lim, et al., ibid., Sauri, et al., ibid., and Kim, et al., ibid areincorporated by reference herein in their entireties.

A preferred Dirofilaria or Brugia TPx-2 protein of the present inventionis a compound that when administered to an animal in an effectivemanner, is capable of protecting that animal from disease caused by aparasitic helminth. In accordance with the present invention, theability of a TPx-2 protein of the present invention to protect an animalfrom disease by a parasitic helminth refers to the ability of thatprotein to, for example, treat, ameliorate or prevent disease caused byparasitic helminths. In one embodiment, a Dirofilaria or Brugia TPx-2protein of the present invention can elicit an immune response(including a humoral and/or cellular immune response) against aparasitic helminth.

Suitable parasites to target include any parasite that is essentiallyincapable of causing disease in an animal administered a Dirofilaria orBrugia TPx-2 protein of the present invention. As such, a parasite totarget includes any parasite that produces a protein having one or moreepitopes that can be targeted by a humoral or cellular immune responseagainst a Dirofilaria or Brugia TPx-2 protein of the present inventionor that can be targeted by a compound that otherwise inhibits parasiteTPx-2 activity, thereby resulting in the decreased ability of theparasite to cause disease in an animal. Preferred parasites to targetinclude parasitic helminths such as nematodes, cestodes, and trematodes,with nematodes being preferred. Preferred nematodes to target includefilariid, ascarid, capillarid, strongylid, strongyloides,trichostrongyle, and trichurid nematodes. Particularly preferrednematodes are those of the genera Acanthocheilonema, Aelurostrongylus,Ancylostoma, Angiostrongylus, Ascaris, Brugia, Bunostomum, Capillaria,Chabertia, Cooperia, Crenosoma, Dictyocaulus, Dioctophyme, Dipetalonema,Diphyllobothrium, Diplydium, Dirofilaria, Dracunculus, Enterobius,Filaroides, Haemonchus, Lagochilascaris, Loa, Mansonella, Muellerius,Nanophyetus, Necator, Nematodirus, Oesophagostomum, Onchocerca,Opisthorchis, Ostertagia, Parafilaria, Paragonimus, Parascaris,Physaloptera, Protostrongylus, Setaria, Spirocerca, Spirometra,Stephanofilaria, Strongyloides, Strongylus, Thelazia, Toxascaris,Toxocara, Trichinella, Trichostrongylus, Trichuris. Uncinaria, andWuchereria. Preferred filariid nematodes include Dirofilaria,Onchocerca, Acanthocheilonema, Brugia, Dipetalonema, Loa, Parafilaria,Setaria, Stephanofilaria and Wuchereria filariid nematodes, with D.immitis and B. malayi being even more preferred.

The present invention also includes mimetopes of Dirofilaria and BrugiaTPx-2 proteins of the present invention. As used herein, a mimetope of aDirofilaria or Brugia TPx-2 protein of the present invention refers toany compound that is able to mimic the activity of such a TPx-2 protein,often because the mimetope has a structure that mimics the particularTPx-2 protein. A mimetope can be, but is not limited to: a peptide thathas been modified to decrease its susceptibility to degradation such asan all-D retro peptide; an anti-idiotypic or catalytic antibody, or afragment thereof; a non-proteinaceous immunogenic portion of an isolatedprotein (e.g., a carbohydrate structure); or a synthetic or naturalorganic molecule, including a nucleic acid. Such a mimetope can bedesigned using computer-generated structures of proteins of the presentinvention. A mimetope can also be obtained by generating random samplesof molecules, such as oligonucleotides, peptides or other organicmolecules, and screening such samples by affinity chromatographytechniques using the corresponding binding partner.

In one embodiment, a Dirofilaria or Brugia TPx-2 protein of the presentinvention is a fusion protein that includes a Dirofilaria or BrugiaTPx-2 protein-containing domain attached to one or more fusion segments.Suitable fusion segments for use with the present invention include, butare not limited to, segments that can: enhance a protein's stability;act as an immunopotentiator to enhance an immune response against aDirofilaria or Brugia TPx-2 protein; or assist purification of aDirofilaria or Brugia TPx-2 protein (e.g., by affinity chromatography).A suitable fusion segment can be a domain of any size that has thedesired function (e.g., imparts increased stability, imparts increasedimmunogenicity to a protein, or simplifies purification of a protein).Fusion segments can be joined to the amino or carboxyl termini of aDirofilaria TPx-2 protein or a Brugia TPx-2 protein-containing domain,and can be susceptible to cleavage in order to enable straight-forwardrecovery of a Dirofilaria or Brugia TPx-2 protein. A fusion protein ispreferably produced by culturing a recombinant cell transformed with afusion nucleic acid molecule that encodes a protein including a fusionsegment attached to either the carboxyl or amino terminal end of a TPx-2protein-containing domain. Preferred fusion segments include a metalbinding domain (e.g., a poly-histidine segment); an immunoglobulinbinding domain (e.g., Protein A; Protein G; T cell; B cell; Fc receptoror complement protein antibody-binding domains); a sugar binding domain(e.g., a maltose binding domain); and/or a "tag" domain (e.g., at leasta portion of β-galactosidase, a strep tag peptide, a T7-tag peptide, aFLAG™ peptide, or other domain that can be purified using compounds thatbind to the domain, such as monoclonal antibodies). More preferredfusion segments include metal binding domains, such as a poly-histidinesegment; a maltose binding domain; a strep tag peptide, such as thatavailable from Biometra® in Tampa, Fla.; and an S10 peptide. An exampleof a particularly preferred fusion protein of the present invention isPHIS-PDiTPx2₂₃₅, production of which is disclosed herein, andPHIS-PBmTPx2₂₃₅, which can be produced in a similar manner.

In another embodiment, a Dirofilaria or Brugia TPx-2 protein of thepresent invention also includes at least one additional protein segmentthat is capable of protecting an animal from one or more diseases. Sucha multivalent protective protein can be produced by culturing a celltransformed with a nucleic acid molecule comprising two or more nucleicacid domains joined together in such a manner that the resulting nucleicacid molecule is expressed as a multivalent protective compoundcontaining at least two protective compounds, or portions thereof,capable of protecting an animal from diseases caused, for example, by atleast one infectious agent.

Examples of multivalent protective compounds include, but are notlimited to, a Dirofilaria or Brugia TPx-2 protein of the presentinvention attached to one or more compounds protective against one ormore other infectious agents, particularly an agent that infects humans,cats, dogs, ferrets, cattle or horses, such as, but not limited to:viruses (e.g., adenoviruses, caliciviruses, coronaviruses, distemperviruses, hepatitis viruses, herpesviruses, immunodeficiency viruses,infectious peritonitis viruses, leukemia viruses, oncogenic viruses,panleukopenia viruses, papilloma viruses, parainfluenza viruses,parvoviruses, rabies viruses, and reoviruses, as well as othercancer-causing or cancer-related viruses); bacteria (e.g., Actinomyces,Bacillus, Bacteroides, Bordetella, Bartonella, Borrelia, Brucella,Campylobacter, Capnocytophaga, Clostridium, Corynebacterium, Coxiella,Dermatophilus, Enterococcus, Ehrlichia, Escherichia, Francisella,Fusobacterium, Haemobartonella, Helicobacter, Klebsiella, L-formbacteria, Leptospira, Listeria, Mycobacteria, Mycoplasma, Neorickettsia,Nocardia, Pasteurella, Peptococcus, Peptostreptococcus, Proteus,Pseudomonas, Rickettsia, Rochalimaea, Salmonella, Shigella,Staphylococcus, Streptococcus, and Yersinia; fungi and fungal-relatedmicroorganisms (e.g., Absidia, Acremonium, Alternaria, Aspergillus,Basidiobolus, Bipolaris, Blastomyces, Candida, Chlamydia, Coccidioides,Conidiobolus, Cryptococcus, Curvalaria, Epidermophyton, Exophiala,Geotrichum, Histoplasma, Madurella, Malassezia, Microsporum, Moniliella,Mortierella, Mucor, Paecilomyces, Penicillium, Phialemonium,Phialophora, Prototheca, Pseudallescheria, Pseudomicrodochium, Pythium,Rhinosporidium, Rhizopus, Scolecobasidium, Sporothrix, Stemphylium,Trichophyton, Trichosporon, and Xylohypha; and other parasites (e.g.,Babesia, Balantidium, Besnoitia, Cryptosporidium, Eimeria,Encephalitozoon, Entamoeba, Giardia, Hammondia, Hepatozoon, Isospora,Leishmania, Microsporidia, Neospora, Nosema, Pentatrichomonas,Plasmodium, Pneumocystis, Sarcocystis, Schistosoma, Theileria,Toxoplasma, and Trypanosoma, as well as helminth parasites, such asthose disclosed herein). In one embodiment, a Dirofilaria or BrugiaTPx-2 protein of the present invention is attached to one or moreadditional compounds protective against heartworm disease,elephantiasis, or hydrocele. In another embodiment, one or moreprotective compounds, such as those listed above, can be included in amultivalent vaccine comprising a Dirofilaria or Brugia TPx-2 protein ofthe present invention and one or more other protective molecules asseparate compounds.

In one embodiment, a preferred isolated TPx-2 protein of the presentinvention is a protein encoded by a nucleic acid molecule comprising atleast a portion of nDiTPx2₈₁₈, nDiTPx2₈₀₂, nDiTPx2₇₀₉, nDiTPx2₇₀₅,nDiTPx2₇₃₆, nBmTPx2₇₃₆, and nBmTPx2₇₀₅, or by an allelic variant of anyof these nucleic acid molecules. Also preferred is an isolated TPx-2protein encoded by a nucleic acid molecule having the nucleic acidsequence SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:11 or SEQ ID NO:20; or by an allelic variant of a nucleic acidmolecule having any of these sequences.

Translation of SEQ ID NO:1, the coding strand of nucleic acid moleculenDiTPx2₈₀₂, yields an apparently full-length D. immitis TPx-2 protein ofabout 235 amino acids, referred to herein as PDiTPx2₂₃₅, the amino acidsequence of which is represented by SEQ ID NO:2, assuming an openreading frame having an initiation (start) codon spanning fromnucleotide 13 through nucleotide 15 of SEQ ID NO:1 and a termination(stop) codon spanning from nucleotide 718 through nucleotide 720 of SEQID NO:1. The coding region encoding PDiTPx2₂₃₅, not including the stopcodon, is represented by nucleic acid molecule nDiTPx2₇₀₅, having thenucleic acid sequence represented by SEQ ID NO:4 (the coding strand) andSEQ ID NO:5 (the complementary strand). The deduced amino acid sequenceSEQ ID NO:2 encodes a protein having a molecular weight of about 26.5kilodaltons (kD) and a predicted pI of about 5.29. In addition, SEQ IDNO:2 includes a Cys residue at position 49. While not being bound bytheory, this Cys residue is most likely the active site of PDiTPx2₂₃₅.

Comparison of amino acid sequence SEQ ID NO:2 (i.e., the amino acidsequence of PDiTPx2₂₃₅) with amino acid sequences reported in GenBank™indicates that SEQ ID NO:2 shares similarity with TPx proteins ofeukaryotic origin. The highest scoring match, i.e., about 86% identityover a region spanning from about amino acid 1 through about amino acid235 of SEQ ID NO:2, was found between SEQ ID NO:2 and an O. volvulusadult TPx protein (GenBank™ Accession No. P52570). SEQ ID NO:2 was alsoaligned with the amino acid sequence of the D. immitis TPx-1 proteindisclosed as SEQ ID NO:2 in copending U.S. patent application Ser. No.08/602,010, ibid. Optimal alignment revealed that a region of SEQ IDNO:2, spanning from about amino acid 1 through about amino acid 235, hadonly about 27% identity with the D. immitis TPx-1 amino acid sequence,confirming that these proteins are only distantly related.

Translation of SEQ ID NO:8, the coding strand of nucleic acid moleculenBmTPx2₇₃₆, yields an apparently full-length B. malayi TPx-2 protein ofabout 235 amino acids, referred to herein as PBmTPx2₂₃₅, the amino acidsequence of which is represented by SEQ ID NO:9, assuming an openreading frame having an initiation codon spanning from nucleotide 29through nucleotide 31 of SEQ ID NO:8 and a termination codon spanningfrom nucleotide 734 through nucleotide 736 of SEQ ID NO:8. The codingregion encoding PBmTPx2₂₃₅, not including the stop codon, is representedby nucleic acid molecule nBmTPx2₇₀₅, having the nucleic acid sequencerepresented by SEQ ID NO:11 (the coding strand) and SEQ ID NO:12 (thecomplementary strand). The deduced amino acid sequence SEQ ID NO:9suggests a protein having a molecular weight of about 26.4 kD and apredicted pI of about 5.29. In addition SEQ ID NO:9 includes a Cysresidue at position 49. While not being bound by theory, this Cysresidue is most likely the active site of PBmTPx2₂₃₅.

Comparison of amino acid sequence SEQ ID NO:9 (i.e., the amino acidsequence of PBmTPx2₂₃₅) with amino acid sequences reported in GenBank™indicates that SEQ ID NO:9 shares similarity with TPx proteins ofeukaryotic origin. The highest scoring match, i.e., about 81% identityover a region extending from about amino acid 1 through about amino acid235 of SEQ ID NO:9, was found between SEQ ID NO:9 and an O. volvulusadult TPx protein (GenBank™ Accession No. P52570). SEQ ID NO:9 was alsocompared to the D. immitis TPx-2 amino acid sequence, SEQ ID NO:2 of thepresent invention. These sequences showed about 85% identity spanningfrom amino acid 1 through about amino acid 235 of both sequences. SEQ IDNO:9 was also aligned with the amino acid sequence of the D. immitisTPx-1 protein disclosed as SEQ ID NO:2 in copending U.S. patentapplication Ser. No. 08/602,010, ibid. Optimal alignment revealed that aregion of SEQ ID NO:9, spanning from about amino acid 1 through aboutamino acid 235, had only about 27% identity with the D. immitis TPx-1amino acid sequence SEQ ID NO:2, confirming that these proteins are onlydistantly related.

A preferred TPx-2 protein of the present invention comprises a proteinthat is at least about 90%, and preferably at least about 95% identicalto PDiTPx2₂₃₅ or PBmTPx2₂₃₅. More preferred is a TPx-2 proteincomprising PDiTPx2₂₃₅ or PBmTPx2₂₃₅ ; or a protein encoded by an allelicvariant of a nucleic acid molecule encoding a protein comprisingPDiTPx2₂₃₅ or PBmTPx2₂₃₅.

Also preferred is a TPx-2 protein comprising an amino acid sequence thatis at least about 90%, and preferably at least about 95% identical toamino acid sequence SEQ ID NO:2 or SEQ ID NO:9.

A particularly preferred Dirofilaria TPx-2 protein of the presentinvention comprises amino acid sequence SEQ ID NO:2, including, but notlimited to, a TPx-2 protein consisting of amino acid sequence SEQ IDNO:2, a fusion protein or a multivalent protein; or a protein encoded byan allelic variant of a nucleic acid molecule encoding a protein havingamino acid sequence SEQ ID NO:2. A particularly preferred Brugia TPx-2protein of the present invention comprises amino acid sequence SEQ IDNO:9, including, but not limited to, a TPx-2 protein consisting of SEQID NO:9, a fusion protein or a multivalent protein; and a proteinencoded by an allelic variant of a nucleic acid molecule encoding aprotein having amino acid sequence SEQ ID NO:9.

Another embodiment of the present invention is an isolated nucleic acidmolecule comprising a Dirofilaria TPx-2 nucleic acid molecule or aBrugia TPx-2 nucleic acid molecule. The identifying characteristics ofsuch a nucleic acid molecule is heretofore described. A nucleic acidmolecule of the present invention can include an isolated naturalDirofilaria or Brugia TPx-2 gene or a homolog thereof, the latter ofwhich is described in more detail below. A nucleic acid molecule of thepresent invention can include one or more regulatory regions, afull-length or a partial coding region, or a combination thereof. Theminimal size of a nucleic acid molecule of the present invention is asize sufficient to allow the formation of a stable hybrid (i.e.,hybridization under stringent hybridization conditions) with thecomplementary sequence of another nucleic acid molecule. As such, theminimal size of a TPx-2 nucleic acid molecule of the present inventionis from about 12 to about 18 nucleotides in length. A preferred TPx-2nucleic acid molecule includes a D. immitis TPx-2 nucleic acid moleculeor a B. malayi TPx-2 nucleic acid molecule.

In accordance with the present invention, an isolated nucleic acidmolecule is a nucleic acid molecule that has been removed from itsnatural milieu (i.e., that has been subject to human manipulation) andcan include DNA, RNA, or derivatives of either DNA or RNA. As such,"isolated" does not reflect the extent to which the nucleic acidmolecule has been purified. An isolated Dirofilaria or Brugia TPx-2nucleic acid molecule of the present invention can be isolated from itsnatural source or produced using recombinant DNA technology (e.g.,polymerase chain reaction (PCR) amplification or cloning) or chemicalsynthesis. Isolated Dirofilaria or Brugia TPx-2 nucleic acid moleculescan include, for example, natural allelic variants and nucleic acidmolecules modified by nucleotide insertions, deletions, substitutions,or inversions in a manner such that the modifications do notsubstantially interfere with the nucleic acid molecule's ability toencode a TPx-2 protein of the present invention.

A Dirofilaria or Brugia TPx-2 nucleic acid molecule homolog can beproduced using a number of methods known to those skilled in the art.See, for example, Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Labs Press; Sambrook et al., ibid., isincorporated by reference herein in its entirety. For example, a nucleicacid molecule can be modified using a variety of techniques including,but not limited to, classic mutagenesis and recombinant DNA techniquessuch as site-directed mutagenesis, chemical treatment, restrictionenzyme cleavage, ligation of nucleic acid fragments, PCR amplification,synthesis of oligonucleotide mixtures and ligation of mixture groups to"build" a mixture of nucleic acid molecules, and combinations thereof.

A nucleic acid molecule homolog can be selected by hybridization with aDirofilaria or Brugia TPx-2 nucleic acid molecule or by screening thefunction of a protein encoded by the nucleic acid molecule (e.g.,ability to elicit an immune response against at least one epitope of aDirofilaria or Brugia TPx-2 protein, the ability to bind to immuneserum, or thioredoxin peroxidase activity).

An isolated nucleic acid molecule of the present invention can include anucleic acid sequence that encodes a Dirofilaria or Brugia TPx-2 proteinof the present invention, examples of such proteins being disclosedherein. Although the phrase "nucleic acid molecule" primarily refers tothe physical nucleic acid molecule and the phrase "nucleic acidsequence" primarily refers to the sequence of nucleotides on the nucleicacid molecule, the two phrases can be used interchangeably, especiallywith respect to a nucleic acid molecule, or a nucleic acid sequence,being capable of encoding a Dirofilaria or Brugia TPx-2 protein.

A preferred nucleic acid molecule of the present invention, whenadministered to an animal, is capable of protecting that animal fromdisease caused by a parasitic helminth. As will be disclosed in moredetail below, such a nucleic acid molecule can be, or can encode, anantisense RNA, a molecule capable of triple helix formation, a ribozyme,or other nucleic acid-based drug compound. In additional embodiments, anucleic acid molecule of the present invention can encode a protectiveprotein (e.g., a TPx-2 protein of the present invention), the nucleicacid molecule being delivered to the animal, for example, by directinjection (i.e, as a genetic vaccine) or in a vehicle such as arecombinant virus vaccine or a recombinant cell vaccine.

Comparison of nucleic acid sequence SEQ ID NO:4 (i.e., the nucleic acidsequence of nDiTPx2₇₀₅) and SEQ ID NO:11 (i.e., the nucleic acidsequence of nBmTPx2₇₀₅) with nucleic acid sequences reported in GenBank™indicates that both SEQ ID NO:4 and SEQ ID NO:11 are similar to genesencoding TPx proteins of eukaryotic origin. A region of SEQ ID NO:4spanning from about nucleotide 1 through about nucleotide 705 was foundto share about 86% identity with the coding region of an O. volvulusadult TPx cDNA molecule, GenBank™ Accession No. U31052. A region of SEQID NO:11 spanning from about nucleotide 1 through about nucleotide 705was found to share about 84% identity with the coding region of an O.volvulus adult TPx cDNA molecule, GenBank™ Accession No. U31052. SEQ IDNO:4 and SEQ ID NO:11 of the present invention were also aligned withthe nucleotide sequence of the D. immitis TPx-1 coding region disclosedas SEQ ID NO:4 in copending U.S. patent application Ser. No. 08/602,010,ibid. Optimal alignments revealed that a region of SEQ ID NO:4, spanningfrom about nucleotide 1 through about nucleotide 705, shared about 46%identity with the D. immitis TPx-1 coding region nucleotide sequence,and a region of SEQ ID NO:11, spanning from about nucleotide 1 throughabout nucleotide 705, shared about 48% identity with the D. immitisTPx-1 coding region nucleotide sequence.

In one embodiment, a Dirofilaria or Brugia TPx-2 nucleic acid moleculeof the present invention includes a nucleic acid molecule that is atleast about 90% and preferably at least about 95% identical to nucleicacid molecule nDiTPx2₈₁₈, nDiTPx2₈₀₂, nDiTPx2₇₀₉, nDiTPx2₇₀₅,nDiTPx2₇₃₆, nBmTPx2₇₃₆ and nBmTPx2₇₀₅ ; or an allelic variant of any ofthese nucleic acid molecules. Also preferred is a Dirofilaria or BrugiaTPx-2 nucleic acid molecule comprising a nucleic acid sequence that isat least about 90% and preferably at least about 95% identical tonucleic acid sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7 SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:20 or SEQ ID NO:21; or an allelic variant of anucleic acid molecule having any of these sequences.

Particularly preferred is a TPx-2 nucleic acid molecule comprising allor part of nucleic acid molecule nDiTPx2₈₁₈, nDiTPx2₈₀₂, nDiTPx2₇₀₉,nDiTPx2₇₀₅, nDiTPx2₇₃₆, nBmTPx2₇₃₆, and nBmTPx2₇₀₅ ; or an allelicvariant of any these nucleic acid molecules. Also particularly preferredis a nucleic acid molecule that includes at least a portion of nucleicacid sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:20, or SEQ ID NO:21; or an allelic variant of a nucleic acid moleculehaving any of these nucleic acid sequences. Such a nucleic acid moleculecan include nucleotides in addition to those included in the SEQ ID NOS,such as, but not limited to, nucleotides comprising a full-length gene;or nucleotides comprising a nucleic acid molecule encoding a fusionprotein or a nucleic acid molecule encoding a multivalent protectivecompound.

The present invention also includes a nucleic acid molecule encoding aprotein having at least a portion of SEQ ID NO:2 or SEQ ID NO:9; or anallelic variant of a nucleic acid molecule encoding a protein having atleast a portion of SEQ ID NO:2 or SEQ ID NO:9. The present inventionfurther includes a nucleic acid molecule that has been modified toaccommodate codon usage properties of a cell in which such a nucleicacid molecule is to be expressed.

Knowing the nucleic acid sequences of certain Dirofilaria or BrugiaTPx-2 nucleic acid molecules of the present invention allows one skilledin the art to, for example, (a) make copies of those nucleic acidmolecules, (b) obtain nucleic acid molecules including at least aportion of such nucleic acid molecules (e.g., nucleic acid moleculesincluding full-length genes, full-length coding regions, regulatorycontrol sequences, truncated coding regions), and (c) obtain otherDirofilaria or Brugia TPx-2 nucleic acid molecules. Such nucleic acidmolecules can be obtained in a variety of ways including screeningappropriate expression libraries with antibodies of the presentinvention; traditional cloning techniques using oligonucleotide probesof the present invention to screen appropriate libraries; and PCRamplification of appropriate libraries or DNA using oligonucleotideprimers of the present invention. Preferred libraries to screen or fromwhich to amplify nucleic acid molecules include Dirofilaria or BrugiaL3, L4 or adult cDNA libraries as well as genomic DNA libraries.Similarly, preferred DNA sources from which to amplify nucleic acidmolecules include Dirofilaria or Brugia L3, L4 or adult first-strandcDNA syntheses and genomic DNA. Techniques to clone and amplify genesare disclosed, for example, in Sambrook et al., ibid.

The present invention also includes a nucleic acid molecule that is anoligonucleotide capable of hybridizing, under stringent hybridizationconditions, with complementary regions of other, preferably longer,nucleic acid molecules of the present invention such as those comprisingDirofilaria or Brugia TPx-2 nucleic acid molecules; or withcomplementary regions of other parasitic helminth TPx-2 nucleic acidmolecules. An oligonucleotide of the present invention can be RNA, DNA,or derivatives of either. The minimum size of such an oligonucleotide isthe size required for formation of a stable hybrid between theoligonucleotide and a complementary sequence on another nucleic acidmolecule. A preferred oligonucleotide of the present invention has amaximum size of about 100 nucleotides. The present invention includesoligonucleotides that can be used as, for example, probes to identifynucleic acid molecules, primers to produce nucleic acid molecules, ortherapeutic reagents to inhibit Dirofilaria or Brugia TPx-2 proteinproduction or activity (e.g., as antisense-, triplex formation-,ribozyme- and/or RNA drug-based reagents). The present invention alsoincludes the use of such oligonucleotides to protect animals fromdisease using one or more of such technologies. Appropriateoligonucleotide-containing therapeutic compositions can be administeredto an animal using techniques known to those skilled in the art.

Another embodiment of the present invention includes a recombinantvector, which includes at least one isolated nucleic acid molecule ofthe present invention inserted into any vector capable of delivering thenucleic acid molecule into a host cell. Such a vector containsheterologous nucleic acid sequences, that is, nucleic acid sequencesthat are not naturally found adjacent to nucleic acid molecules of thepresent invention, and that preferably are derived from a species otherthan the species from which the nucleic acid molecule(s) are derived.The vector can be either RNA or DNA, either prokaryotic or eukaryotic,and typically is a virus or a plasmid. Recombinant vectors can be usedto clone, sequence, or otherwise manipulate a Dirofilaria or BrugiaTPx-2 nucleic acid molecule of the present invention.

One type of recombinant vector, referred to herein as a recombinantmolecule, comprises a nucleic acid molecule of the present inventionoperatively linked to an expression vector. The phrase "operativelylinked" refers to insertion of a nucleic acid molecule into anexpression vector in a manner such that the molecule is able to beexpressed when transformed into a host cell. As used herein, anexpression vector is a DNA or RNA vector that is capable of transforminga host cell and of effecting expression of a specified nucleic acidmolecule. Preferably, the expression vector is also capable ofreplicating within the host cell. An expression vector can be eitherprokaryotic or eukaryotic, and is typically a virus or a plasmid. Anexpression vector of the present invention includes any vector thatfunctions (i.e., directs gene expression) in a recombinant cell of thepresent invention, including in a bacterial, fungal, parasite, insect,other animal, or plant cell. A preferred expression vector of thepresent invention can direct gene expression in a bacterial, yeast,helminth or other parasite, insect or mammalian cell, or more preferablyin a cell type disclosed herein.

In particular, an expression vector of the present invention containsregulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the recombinant cell andthat control the expression of a nucleic acid molecule of the presentinvention. In particular, a recombinant molecule of the presentinvention includes transcription control sequences. Transcriptioncontrol sequences are sequences which control the initiation,elongation, and termination of transcription. Particularly importanttranscription control sequences are those which control transcriptioninitiation, such as promoter, enhancer, operator and repressorsequences. A suitable transcription control sequence includes anytranscription control sequence that can function in at least one of therecombinant cells of the present invention. A variety of suchtranscription control sequences are known to those skilled in the art.Preferred transcription control sequences include those which functionin bacterial, yeast, helminth or other parasite, insect or mammaliancells, such as, but not limited to, tac, lac, trp, trc, oxy-pro,omp/lpp, rrnB, bacteriophage lambda (such as lambda p_(L) and lambdaP_(R) and fusions that include such promoters), bacteriophage T7, T7lac,bacteriophage T3, bacteriophage SP6, bacteriophage SP01,metallothionein, alphamating factor, Pichia alcohol oxidase, alphavirussubgenomic promoters (such as Sindbis virus subgenomic promoters),antibiotic resistance gene, baculovirus, Heliothis zea insect virus,vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus,adenovirus, cytomegalovirus (such as immediate early promoters),picornavirus, simian virus 40, retrovirus, actin, retroviral longterminal repeat, Rous sarcoma virus, heat shock, phosphate or nitratetranscription control sequences; as well as other sequences capable ofcontrolling gene expression in prokaryotic or eukaryotic cells.Additional suitable transcription control sequences includetissue-specific promoters and enhancers as well as lymphokine-induciblepromoters (e.g., promoters inducible by interferons or interleukins).Transcription control sequences of the present invention can alsoinclude naturally occurring transcription control sequences naturallyassociated with parasitic helminths, such as D immitis or B. malayi.

Suitable and preferred nucleic acid molecules to include in arecombinant vector of the present invention are as disclosed herein. Apreferred nucleic acid molecule to include in a recombinant vector, andparticularly in a recombinant molecule, includes nDiTPx2₈₁₈, nDiTPx2₈₀₂,nDiTPx2₇₀₉, nDiTPx2₇₀₅, nDiTPx2₇₃₆, nBmTPx2₇₃₆, and nBmTPx2₇₀₅.Particularly preferred recombinant molecules of the present inventioninclude pβgal-nDiTPx2₈₀₂ and pTrc-nDiTPx2₇₀₉, the production of whichare described in the Examples section, and pTrc-nBmTPx2₇₀₉, which can beproduced in a similar manner.

A recombinant molecule of the present invention may also (a) contain asecretory signal (i.e., a signal segment nucleic acid sequence) toenable an expressed TPx-2 protein of the present invention to besecreted from the cell that produces the protein or (b) contain a fusionsequence which leads to the expression of a nucleic acid molecule of thepresent invention as a fusion protein. Examples of suitable signalsegments include any signal segment capable of directing the secretionof a protein of the present invention. Preferred signal segmentsinclude, but are not limited to, native Dirofilaria or Brugia, tissueplasminogen activator (t-PA), interferon, interleukin, growth hormone,histocompatibility and viral envelope glycoprotein signal segments.Suitable fusion segments encoded by fusion segment nucleic acids aredisclosed herein. In addition, a nucleic acid molecule of the presentinvention can be joined to a fusion segment that directs the encodedprotein to the proteosome, such as a ubiquitin fusion segment. Aeukaryotic recombinant molecule may also include intervening and/oruntranslated sequences surrounding and/or within the nucleic acidsequence of the nucleic acid molecule of the present invention.

Another embodiment of the present invention includes a recombinant cellcomprising a host cell transformed with one or more recombinantmolecules of the present invention. Transformation of a nucleic acidmolecule into a cell can be accomplished by any method by which anucleic acid molecule can be inserted into the cell. Transformationtechniques include, but are not limited to, transfection,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. A recombinant cell may remain unicellular or may grow into atissue, organ, or a multicellular organism. Transformed nucleic acidmolecules of the present invention can remain extrachromosomal or canintegrate into one or more sites within a chromosome of the transformed(i.e., recombinant) cell in such a manner that their ability to beexpressed is retained. Preferred nucleic acid molecules with which totransform a cell include TPx-2 nucleic acid molecules disclosed herein.Particularly preferred nucleic acid molecules with which to transform acell include nDiTPx2₈₁₈, nDiTPx2₈₀₂, nDiTPx2₇₀₅, nDiTPx2₇₃₆, nBmTPx2₇₃₆,and nBmTPx2₇₀₅.

Suitable host cells to transform include any cell that can betransformed with a nucleic acid molecule of the present invention. Hostcells can be either untransformed cells or cells that are alreadytransformed with at least one nucleic acid molecule (e.g., nucleic acidmolecules encoding one or more proteins of the present invention orencoding other proteins useful in the production of multivalentvaccines). A recombinant cell of the present invention can beendogenously (i.e., naturally) capable of producing a Dirofilaria orBrugia TPx-2 protein of the present invention or can be capable ofproducing such a protein after being transformed with at least onenucleic acid molecule of the present invention. A host cell of thepresent invention can be any cell capable of producing at least oneprotein of the present invention, and can be a bacterial, fungal(including yeast), parasite (including helminth, protozoa andectoparasite), other insect, other animal or plant cell. Preferred hostcells include bacterial, mycobacterial, yeast, helminth, insect andmammalian cells. More preferred host cells include Salmonella,Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera,Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells(Madin-Darby Canine Kidney cells), CRFK cells (Crandell Feline Kidneycells), BSC-1 cells (African monkey kidney cell line used, for example,to culture poxviruses), COS (e.g., COS-7) cells, and Vero cells.Particularly preferred host cells are Escherichia coli, including E.coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium,including attenuated strains such as UK-1_(x) 3987 and SR-11_(x) 4072;Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCK cells; CRFKcells; BSC-1 cells; COS cells; Vero cells; and non-tumorigenic mousemyoblast G8 cells (e.g., ATCC CRL 1246). Additional appropriatemammalian cell hosts include other kidney cell lines, other fibroblastcell lines (e.g., human, murine or chicken embryo fibroblast celllines), myeloma cell lines, Chinese hamster ovary cells, mouse NIH/3T3cells, LMTK³¹ cells and/or HeLa cells. In one embodiment, the proteinsmay be expressed as heterologous proteins in myeloma cell linesemploying immunoglobulin promoters.

A recombinant cell of the present invention includes any celltransformed with at least one of any nucleic acid molecule of thepresent invention. Suitable and preferred nucleic acid molecules as wellas suitable and preferred recombinant molecules with which to transformsuch a cell are disclosed herein. Particularly preferred recombinantcells include E. coli:pβgal-nDiTPx2₈₀₂ and E. coli:pTrc-nDiTPx2₇₀₉, theproduction of which is disclosed herein, and E. coli:pTrc-nBmTPx2₇₀₉,which can be produced in a similar manner.

In one embodiment, a recombinant cell of the present invention can beco-transformed with a recombinant molecule including a Dirofilaria orBrugia TPx-2 nucleic acid molecule encoding a protein of the presentinvention and a nucleic acid molecule encoding another protectivecompound, as disclosed herein (e.g., to produce multivalent vaccines).

Recombinant DNA technologies can be used to improve expression of atransformed nucleic acid molecule by manipulating, for example, thenumber of copies of the nucleic acid molecule within a host cell, theefficiency with which that nucleic acid molecule is transcribed, theefficiency with which the resultant transcript is translated, and theefficiency of post-translational modifications. Recombinant techniquesuseful for increasing the expression of a nucleic acid molecule of thepresent invention include, but are not limited to, operatively linkingthe nucleic acid molecule to a high-copy number plasmid, integration ofthe nucleic acid molecule into one or more host cell chromosomes,addition of vector stability sequences to a plasmid, substitution ormodification of transcription control signals (e.g., promoters,operators, enhancers), substitution or modification of translationalcontrol signals (e.g., ribosome binding sites, Shine-Dalgamo sequences,or Kozak sequences), modification of a nucleic acid molecule of thepresent invention to correspond to the codon usage of the host cell,deletion of sequences that destabilize transcripts, and the use ofcontrol signals that temporally separate recombinant cell growth fromrecombinant enzyme production during fermentation. The activity of anexpressed recombinant protein of the present invention may be improvedby fragmenting, modifying, or derivatizing a nucleic acid moleculeencoding such a protein.

Isolated Dirofilaria or Brugia TPx-2 proteins of the present inventioncan be produced in a variety of ways, including production and recoveryof natural proteins, production and recovery of recombinant proteins,and chemical synthesis of the proteins. In one embodiment, an isolatedprotein of the present invention is produced by culturing a cell capableof expressing the protein under conditions effective to produce theprotein, and recovering the protein. A preferred cell to culture is arecombinant cell of the present invention. Effective culture conditionsinclude, but are not limited to, effective media, bioreactor,temperature, pH and oxygen conditions that permit protein production. Aneffective medium refers to any medium in which a cell is cultured toproduce a Dirofilaria or Brugia TPx-2 protein of the present invention.Such a medium typically comprises an aqueous base having assimilablecarbon, nitrogen and phosphate sources, and appropriate salts, minerals,metals and other nutrients, such as vitamins. Cells of the presentinvention can be cultured in conventional fermentation bioreactors,shake flasks, test tubes, microtiter dishes, and petri plates. Culturingcan be carried out at a temperature, pH and oxygen content appropriatefor a given recombinant cell. Such culturing conditions are within theexpertise of one of ordinary skill in the art. Examples of suitableconditions are included in the Examples section.

Depending on the vector and host system used for production, a resultantprotein of the present invention may either remain within therecombinant cell; be secreted into the fermentation medium; be secretedinto a space between two cellular membranes, such as the periplasmicspace in E. coli; or be retained on the outer surface of a cell or viralmembrane.

The phrase "recovering the protein", as well as similar phrases, referto collecting the whole fermentation medium containing the protein andneed not imply additional steps of separation or purification. Proteinsof the present invention can be purified using a variety of standardprotein purification techniques, such as, but not limited to, affinitychromatography, ion exchange chromatography, filtration,electrophoresis, hydrophobic interaction chromatography, gel filtrationchromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization.Proteins of the present invention are preferably retrieved in"substantially pure" form. As used herein, "substantially pure" refersto a purity that allows for the effective use of the protein as atherapeutic composition or diagnostic. A therapeutic composition foranimals, for example, should exhibit no substantial toxicity andpreferably should be capable of stimulating the production of antibodiesin a treated animal.

The present invention also includes isolated (i.e., removed from theirnatural milieu) antibodies that selectively bind to a Dirofilaria orBrugia TPx-2 protein of the present invention or a mimetope thereof(e.g., anti-Dirofilaria or Brugia TPx-2 antibodies). As used herein, theterm "selectively binds to" a TPx-2 protein refers to the ability of anantibody of the present invention to preferentially bind to specifiedproteins and mimetopes thereof of the present invention. Binding can bemeasured using a variety of methods standard in the art including enzymeimmunoassays (e.g., ELISA), immunoblot assays, etc. See, for example,Sambrook et al., ibid., and Harlow, et al., 1988, Antibodies, aLaboratory Manual, Cold Spring Harbor Labs Press; Harlow et al., ibid.,is incorporated by reference herein in its entirety. An anti-parasitichelminth TPx-2 antibody preferably selectively binds to a Dirofilaria orBrugia TPx-2 protein in such a way as to reduce the activity of thatprotein.

Isolated antibodies of the present invention can include antibodies inserum, or antibodies that have been purified to varying degrees.Antibodies of the present invention can be polyclonal or monoclonal,functional equivalents such as antibody fragments andgenetically-engineered antibodies, including single chain antibodies orchimeric antibodies that can bind to more than one epitope.

A preferred method to produce antibodies of the present inventionincludes (a) administering to an animal an effective amount of aprotein, peptide or mimetope thereof of the present invention to producethe antibodies and (b) recovering the antibodies. In another method,antibodies of the present invention are produced recombinantly usingtechniques as heretofore disclosed to produce TPx-2 proteins of thepresent invention. Antibodies raised against defined proteins ormimetopes can be advantageous because such antibodies are notsubstantially contaminated with antibodies against other substances thatmight otherwise cause interference in a diagnostic assay or side effectsif used in a therapeutic composition.

Antibodies of the present invention have a variety of potential usesthat are within the scope of the present invention. For example, suchantibodies can be used (a) as therapeutic compounds to passivelyimmunize an animal in order to protect the animal from parasitichelminths susceptible to treatment by such antibodies, (b) as reagentsin assays to detect infection by such helminths or (c) as tools toscreen expression libraries or to recover desired proteins of thepresent invention from a mixture of proteins and other contaminants.Furthermore, antibodies of the present invention can be used to targetcytotoxic agents to parasitic helminths of the present invention inorder to directly kill such helminths. Targeting can be accomplished byconjugating (i.e., stably joining) such antibodies to the cytotoxicagents using techniques known to those skilled in the art. Suitablecytotoxic agents are known to those skilled in the art.

One embodiment of the present invention is a therapeutic compositionthat, when administered to an animal in an effective manner, is capableof protecting that animal from disease caused by a parasitic helminth. Atherapeutic composition of the present invention includes at least oneof the following protective compounds: an isolated Dirofilaria or BrugiaTPx-2 protein or a mimetope thereof, an isolated Dirofilaria or BrugiaTPx-2 nucleic acid molecule, an isolated antibody that selectively bindsto a Dirofilaria or Brugia TPx-2 protein, or an inhibitor of TPx-2protein activity identified by its ability to inhibit Dirofilaria orBrugia TPx-2 activity. As used herein, a protective compound refers to acompound that, when administered to an animal in an effective manner, isable to treat, ameliorate, or prevent disease caused by a parasitichelminth. Preferred helminths to target are heretofore disclosed.Examples of proteins, nucleic acid molecules, antibodies and inhibitorsof the present invention are disclosed herein.

The present invention also includes a therapeutic composition comprisingat least one Dirofilaria or Brugia TPx-2-based compound of the presentinvention in combination with at least one additional compoundprotective against one or more infectious agents. Examples of suchcompounds and infectious agents are disclosed herein.

A therapeutic composition of the present invention can be administeredto any animal susceptible to such therapy, preferably to mammals, andmore preferably to dogs, cats, humans, ferrets, horses, cattle, sheepand other pets, work animals, economic food animals, or zoo animals.Preferred animals to protect against heartworm disease include dogs,cats, humans and ferrets, with dogs and cats being particularlypreferred. The preferred animal to protect against elephantiasis andhydrocele is humans.

In one embodiment, a therapeutic composition of the present inventioncan be administered to the vector in which the parasitic helminthdevelops, such as to a mosquito, in order to prevent the spread of D.immitis to the definitive mammalian host. Such administration could beorally or by developing transgenic vectors capable of producing at leastone therapeutic composition of the present invention. In anotherembodiment, a vector, such as a mosquito, can ingest therapeuticcompositions present in the blood of a host that has been administered atherapeutic composition of the present invention.

A therapeutic composition of the present invention can be formulated inan excipient that the animal to be treated can tolerate. Examples ofsuch excipients include water, saline, Ringer's solution, dextrosesolution, Hank's solution, and other aqueous physiologically balancedsalt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil,ethyl oleate, or triglycerides may also be used. Other usefulformulations include suspensions containing viscosity enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipientscan also contain minor amounts of additives, such as substances thatenhance isotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer, and Tris buffer, while examples ofpreservatives include thimerosal, m- or o-cresol, formalin, and benzylalcohol. Standard formulations can either be liquid injectables orsolids which can be taken up in a suitable liquid as a suspension orsolution for injection. Thus, in a non-liquid formulation, the excipientcan comprise dextrose, human serum albumin, preservatives, etc., towhich sterile water or saline can be added prior to administration.

In one embodiment of the present invention, a therapeutic compositioncan include an adjuvant. Adjuvants are agents that are capable ofenhancing the immune response of an animal to a specific antigen.Suitable adjuvants include, but are not limited to, cytokines,chemokines, and compounds that induce the production of cytokines andchemokines (e.g., granulocyte macrophage colony stimulating factor(GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophagecolony stimulating factor (M-CSF), colony stimulating factor (CSF),erythropoietin (EPO), interleukin 2 (IL-2), interleukin-3 (IL-3),interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6),interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10),interleukin 12 (IL-12), interferon gamma, interferon gamma inducingfactor I (IGIF), transforming growth factor beta, RANTES (regulated uponactivation, normal T-cell expressed and presumably secreted), macrophageinflammatory proteins (e.g., MIP-1 alpha and MIP-1 beta), and Leishmaniaelongation initiating factor (LEIF)); bacterial components (e.g.,endotoxins, in particular superantigens, exotoxins and cell wallcomponents); aluminum-based salts; calcium-based salts; silica;polynucleotides; toxoids; serum proteins, viral coat proteins; blockcopolymer adjuvants (e.g., Hunter's Titermax™ adjuvant (Vaxcel™, Inc.Norcross, Ga.), Ribi adjuvants (Ribi ImmunoChem Research, Inc.,Hamilton, Mont.), and saponins and their derivatives (e.g., Quil A(Superfos Biosector A/S, Denmark). Protein adjuvants of the presentinvention can be delivered in the form of the protein themselves or ofnucleic acid molecules encoding such proteins using the methodsdescribed herein.

In one embodiment of the present invention, a therapeutic compositioncan include a carrier. Carriers include compounds that increase thehalf-life of a therapeutic composition in the treated animal. Suitablecarriers include, but are not limited to, polymeric controlled releasevehicles, biodegradable implants, liposomes, bacteria, viruses, othercells, oils, esters, and glycols.

One embodiment of the present invention is a controlled releaseformulation that is capable of slowly releasing a composition of thepresent invention into an animal. As used herein, a controlled releaseformulation comprises a composition of the present invention in acontrolled release vehicle. Suitable controlled release vehiclesinclude, but are not limited to, biocompatible polymers, other polymericmatrices, capsules, microcapsules, microparticles, bolus preparations,osmotic pumps, diffusion devices, liposomes, lipospheres, andtransdermal delivery systems. Other controlled release formulations ofthe present invention include liquids that, upon administration to ananimal, form a solid or a gel in situ. Preferred controlled releaseformulations are biodegradable (i.e., bioerodible).

A preferred controlled release formulation of the present invention iscapable of releasing a composition of the present invention into theblood of the treated animal at a constant rate sufficient to attaintherapeutic dose levels of the composition to protect an animal fromdisease caused by parasitic helminths. The therapeutic composition ispreferably released over a period of time ranging from about 1 to about12 months. A controlled release formulation of the present invention iscapable of effecting a treatment preferably for at least about 1 month,more preferably for at least about 3 months, even more preferably for atleast about 6 months, even more preferably for at least about 9 months,and even more preferably for at least about 12 months.

In order to protect an animal from disease caused by a parasitichelminth, a therapeutic composition of the present invention isadministered to the animal in an effective manner such that thecomposition is capable of protecting that animal from a disease causedby a parasitic helminth. For example, an isolated protein or mimetopethereof is administered in an amount and manner that elicits (i.e.,stimulates) an immune response that is sufficient to protect the animalfrom the disease. Similarly, an antibody of the present invention, whenadministered to an animal in an effective manner, is administered in anamount so as to be present in the animal at a titer that is sufficientto protect the animal from the disease, at least temporarily. Anoligonucleotide nucleic acid molecule of the present invention can alsobe administered in an effective manner, thereby reducing expression ofnative parasitic helminth TPx-2 proteins in order to interfere withdevelopment of the parasitic helminths targeted in accordance with thepresent invention.

Therapeutic compositions of the present invention can be administered toanimals prior to infection in order to prevent infection (i.e., as apreventative vaccine) or can be administered to animals after infectionin order to treat disease caused by the parasitic helminth (i.e., as acurative agent or a therapeutic vaccine).

Acceptable protocols to administer therapeutic compositions in aneffective manner include individual dose size, number of doses,frequency of dose administration, and mode of administration.Determination of such protocols can be accomplished by those skilled inthe art. A suitable single dose is a dose that is capable of protectingan animal from disease when administered one or more times over asuitable time period. For example, a preferred single dose of a protein,mimetope, or antibody therapeutic composition is from about 1 microgram(μg) to about 10 milligrams (mg) of the therapeutic composition perkilogram body weight of the animal. Booster vaccinations can beadministered from about 2 weeks to several years after the originaladministration. Booster administrations preferably are administered whenthe immune response of the animal becomes insufficient to protect theanimal from disease. A preferred administration schedule is one in whichfrom about 10 μg to about 1 mg of the therapeutic composition per kgbody weight of the animal is administered from about one to about twotimes over a time period of from about 2 weeks to about 12 months. Modesof administration can include, but are not limited to, subcutaneous,intradermal, intravenous, intranasal, oral, transdermal, andintramuscular routes.

According to one embodiment, a nucleic acid molecule of the presentinvention can be administered to an animal in a fashion to enableexpression of that nucleic acid molecule into a protective protein orprotective RNA (e.g., an antisense RNA, a ribozyme, a triple helix form,or an RNA drug) in the animal. Nucleic acid molecules can be deliveredto an animal by a variety of methods including, but not limited to, (a)administering a genetic vaccine (e.g., a naked DNA or RNA molecule, suchas is taught, for example, in Wolff et al., 1990, Science 247,1465-1468) or (b) administering a nucleic acid molecule packaged as arecombinant virus vaccine or as a recombinant cell vaccine (i.e., thenucleic acid molecule is delivered by a viral or cellular vehicle).

A genetic (i.e., naked nucleic acid) vaccine of the present inventionincludes a nucleic acid molecule of the present invention and preferablyincludes a recombinant molecule of the present invention that preferablyis replication, or otherwise amplification, competent. A genetic vaccineof the present invention can comprise one or more nucleic acid moleculesof the present invention in the form of, for example, a dicistronicrecombinant molecule. A preferred genetic vaccine includes at least aportion of a viral genome (i.e., a viral vector). Preferred viralvectors include those based on alphaviruses, poxviruses, adenoviruses,herpesviruses, picomaviruses, and retroviruses, with those based onalphaviruses (such as Sindbis or Semliki forest virus), species-specificherpesviruses and poxviruses being particularly preferred. Any suitabletranscription control sequence can be used, including those disclosed assuitable for protein production. Particularly preferred transcriptioncontrol sequences include cytomegalovirus immediate early (preferably inconjunction with Intron-A), Rous sarcoma virus long terminal repeat, andtissue-specific transcription control sequences, as well astranscription control sequences endogenous to viral vectors if viralvectors are used. The incorporation of "strong" poly(A) sequences isalso preferred.

A genetic vaccine of the present invention can be administered in avariety of ways, with intramuscular, subcutaneous, intradermal,transdermal, intranasal and oral routes of administration beingpreferred. A preferred single dose of a genetic vaccine ranges fromabout 1 nanogram (ng) to about 500 μg, depending on the route ofadministration or method of delivery, as can be determined by thoseskilled in the art. Suitable delivery methods include, for example, byinjection, as drops, aerosolized, or topically. Genetic vaccines of thepresent invention can be contained in an aqueous excipient (e.g.,phosphate buffered saline) alone or in a carrier (e.g., lipid-basedvehicles).

A recombinant virus vaccine of the present invention includes arecombinant molecule of the present invention that is packaged in aviral coat and that can be expressed in an animal after administration.Preferably, the recombinant molecule is packaging- orreplication-deficient or encodes an attenuated virus. A number ofrecombinant viruses can be used, including, but not limited to, thosebased on alphaviruses, poxviruses, adenoviruses, herpesviruses,picornaviruses, and retroviruses. Preferred recombinant virus vaccinesare those based on alphaviruses (such as Sindbis virus), raccoonpoxviruses, picornaviruses, and species-specific herpesviruses. Methodsto produce and use a recombinant alphavirus vaccine are disclosed in PCTPublication No. WO 94/17813, by Xiong et al., published Aug. 18, 1994,which is incorporated by reference herein in its entirety.

When administered to an animal, a recombinant virus vaccine of thepresent invention infects cells within the immunized animal and directsthe production of a protective protein or RNA nucleic acid molecule thatis capable of protecting the animal from disease caused by a parasitichelminth as disclosed herein. For example, a recombinant virus vaccinecomprising a Dirofilaria or Brugia TPx-2 nucleic acid molecule of thepresent invention is administered according to a protocol that resultsin the animal producing a sufficient immune response to protect itselffrom heartworm disease. A preferred single dose of a recombinant virusvaccine of the present invention is from about 1×10⁴ to about 1×10⁸virus plaque forming units (pfu) per kilogram body weight of the animal.Administration protocols are similar to those described herein forprotein-based vaccines, with subcutaneous, intramuscular, intranasal andoral administration routes being preferred.

A recombinant cell vaccine of the present invention includes arecombinant cell of the present invention that expresses at least oneprotein of the present invention. Preferred recombinant cells for thisembodiment include Salmonella, E. coli, Listeria, Mycobacterium, S.frugiperda, yeast (including Saccharomyces cerevisiae and Pichiapastoris), BHK, BSC-1, myoblast G8, COS (e.g., COS-7), Vero, MDCK orCRFK recombinant cells. A recombinant cell vaccine of the presentinvention can be administered in a variety of ways but has the advantagethat it can be administered orally, preferably at doses ranging fromabout 10⁸ to about 10¹² cells per kilogram body weight. Administrationprotocols are similar to those described herein for protein-basedvaccines. A recombinant cell vaccine can comprise whole cells, cellsstripped of cell walls or cell lysates.

The efficacy of a therapeutic composition of the present invention toprotect an animal from disease caused by a parasitic helminth can betested in a variety of ways including, but not limited to, detection ofprotective antibodies (using, for example, proteins or mimetopes of thepresent invention), detection of cellular immunity within the treatedanimal, or challenge of the treated animal with the parasitic helminthto determine whether the treated animal is resistant to disease.Challenge studies can include implantation of chambers includingparasitic helminth larvae into the treated animal and/or directadministration of larvae to the treated animal. In one embodiment,therapeutic compositions can be tested in animal models such as mice.Such techniques are known to those skilled in the art.

One preferred embodiment of the present invention is the use ofDirofilaria or Brugia TPx-2 proteins, nucleic acid molecules, antibodiesor inhibitory compounds of the present invention to protect an animalfrom heartworm disease. It is particularly preferred to prevent L3 thatare delivered to the animal by the mosquito intermediate host frommaturing into adult worms. As such, a preferred therapeutic compositionis one that is able to inhibit at least one step in the portion of theparasite's development cycle that includes L3, third molt, L4, fourthmolt, and immature adult prior to entering the circulatory system. Indogs, this portion of the developmental cycle is about 70 days inlength. A particularly preferred therapeutic composition includes a D.immitis TPx-2-based therapeutic composition of the present invention,particularly since TPx-2 is expressed in L3 and L4. Such a compositioncan include a D. immitis TPx-2 nucleic acid molecule, a D. immitis TPx-2protein or a mimetope thereof, anti-D. immitis TPx-2 antibodies, orinhibitors of D. immitis TPx-2 activity. Such therapeutic compositionsare administered to an animal in a manner effective to protect theanimals from heartworm disease. Additional protection may be obtained byadministering additional protective compounds, including other parasitichelminth proteins, nucleic acid molecules, antibodies and inhibitorycompounds, as disclosed herein.

One therapeutic composition of the present invention includes aninhibitor of Dirofilaria or Brugia TPx-2 activity, i.e., a compoundcapable of substantially interfering with the function of a Dirofilariaor Brugia TPx-2 protein, also referred to herein as a TPx-2 inhibitor.In one embodiment, such an inhibitor comprises a compound that interactsdirectly with a TPx-2 protein active site (usually by binding to ormodifying the active site), thereby inhibiting thioredoxin peroxidaseactivity. According to this embodiment, a TPx-2 inhibitor can alsointeract with other regions of a TPx-2 protein to inhibit thioredoxinperoxidase activity, for example, by allosteric interaction. Preferably,a TPx-2 inhibitor of the present invention is identified by its abilityto bind to, or otherwise interact with, a Dirofilaria or Brugia TPx-2protein, thereby inhibiting thioredoxin peroxidase activity of thatprotein. Such a TPx-2 inhibitor is a suitable for inclusion in atherapeutic composition of the present invention as long as the compoundis not harmful to the host animal being treated.

A preferred TPx-2 inhibitor comprises a compound that binds to theactive site cysteine residue of a Dirofilaria or Brugia TPx-2 protein(e.g., Cys-49 of PDiTPx2₂₃₅, or Cys-49 of PBmTPx2₂₃₅), or a compoundthat binds to any other region of a Dirofilaria or Brugia TPx-2 protein(e.g., to an allosteric site) in such a manner that thioredoxinperoxidase activity is inhibited. A TPx-2 inhibitor can comprise a smallinorganic or organic compound (such as N-ethylmaleimide (NEM)), apeptide, a nucleic acid molecule (such as an oligonucleotide), or apeptidomimetic compound.

A TPx-2 inhibitor can be identified using a Dirofilaria or Brugia TPx-2protein of the present invention. As such, one embodiment of the presentinvention is a method to identify a compound capable of inhibiting TPx-2activity of a parasitic helminth susceptible to inhibition by aninhibitor of Dirofilaria or Brugia TPx-2 activity. Such a methodincludes the steps of (a) contacting (e.g., combining, mixing) anisolated Dirofilaria or Brugia TPx-2 protein, preferably a D. immitis ora B. malayi TPx-2 protein, with a putative inhibitory compound underconditions in which, in the absence of the compound, the protein hasTPx-2 activity, and (b) determining if the putative inhibitory compoundinhibits the TPx-2 activity. Putative inhibitory compounds to screeninclude small organic molecules, antibodies (including mimetopesthereof) and substrate analogs. Methods to determine TPx-2 activity areknown to those skilled in the art; see, for example, Rhee, et al.,ibid., Lim, et al., ibid., Sauri, et al., ibid., and Kim, et al., ibid.

The present invention also includes a test kit to identify a compoundcapable of inhibiting TPx-2 activity of a parasitic helminth. Such atest kit includes an isolated Dirofilaria or Brugia TPx-2 protein,preferably a D. immitis or a B. malayi TPx-2 protein, having TPx-2activity, and a means for determining the extent of inhibition of TPx-2activity in the presence of (i.e., effected by) a putative inhibitorycompound. Such compounds are also screened to identify those that aresubstantially not toxic in host animals, e.g., compounds that do notinhibit the activity of mammalian thioredoxin peroxidases.

TPx-2 inhibitors isolated by such a method or test kit can be used toinhibit any parasitic helminth TPx-2 protein that is susceptible to suchan inhibitor. A particularly preferred TPx-2 inhibitor of the presentinvention is capable of protecting an animal from heartworm disease,elephantiasis and/or hydrocele. A therapeutic composition comprising acompound that inhibits TPx-2 activity can be administered to an animalin an effective manner to protect that animal from disease caused by theparasite expressing the targeted TPx-2 enzyme, and preferably to protectthat animal from heartworm disease, elephantiasis or hydrocele.Effective amounts and dosing regimens can be determined using techniquesknown to those skilled in the art.

It is also within the scope of the present invention to use isolatedproteins, mimetopes, nucleic acid molecules and antibodies of thepresent invention as diagnostic reagents to detect infection byparasitic helminths. Such diagnostic reagents can be supplemented withadditional compounds that can detect other phases of the parasite's lifecycle. Methods to use such diagnostic reagents to diagnose parasitichelminth infection are well known to those skilled in the art. Suitableand preferred parasitic helminths to detect are those to whichtherapeutic compositions of the present invention are targeted.Particularly preferred parasitic helminths to detect using diagnosticreagents of the present invention are Dirofilaria and Brugia.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention.

EXAMPLES

It is to be noted that the examples include a number of molecularbiology, microbiology, immunology and biochemistry techniques familiarto those skilled in the art. Disclosure of such techniques can be found,for example, in Sambrook et al., ibid., Ausubel, et al.,1993, CurrentProtocols in Molecular Biology, Greene/Wiley Interscience, New York,N.Y., and related references. Ausubel, et al, ibid. is incorporated byreference herein in its entirety. DNA and protein sequence analyses werecarried out using the PC/GENE™ sequence analysis program (available fromIntelligenetics, Inc., Mountainview, Cailf.) and the Wisconsin Package™Version 9.0 (available from the Genetics Computer Group (GCG), Madison,Wis.). It should also be noted that since nucleic acid sequencingtechnology, and in particular the sequencing of PCR products, is notentirely error-free, that the nucleic acid and deduced protein sequencespresented herein represent apparent nucleic acid sequences of thenucleic acid molecules encoding D. immitis and B. malayi TPx-2 proteinsof the present invention.

Example 1

This example describes the isolation and sequencing of a D. immitisthioredoxin peroxidase type-2 (TPx-2) nucleic acid molecule.

A D. immitis TPx-2 molecule of about 802 nucleotides, denotednDiTPx2₈₀₂, was isolated from a cDNA library by its surprising abilityto encode a protein that selectively bound to at least one component ofimmune serum from a rabbit immunized with a peptide derived from aBrugia malayi transglutaminase protein. Specifically, a D. immitis L4cDNA expression library was constructed in the Uni-ZAP® XR vector(available from Stratagene Cloning Systems, La Jolla, Cailf.), usingStratagene's ZAP-cDNA® Synthesis Kit protocol and L4 mRNAs (i.e., mRNAsisolated from fourth-stage larval D. immitis by standard methods). Thelibrary was immuno-screened using a rabbit anti-Brugia malayitransglutaminase PBmTG20 antiserum, as disclosed Singh, et al., 1995,Int. J. Biochem. Cell Biol. 27, 1285-1291, which is incorporated hereinby reference in its entirety. Briefly, the library was plated onto alawn of E. coli XL1-Blue MRF' (available from Stratagene) at a densityof 25×10³ phage per petri dish (150 mm²) and grown at 37° C. for 4 hr.When plaques were visible, isopropyl-β-D thiogalactoside(IPTG)-impregnated nitrocellulose filters were placed on plates for 3 hrat 37° C. Filters were washed in PBS-Tween (PBS/T) which is 0.01% Mphosphate-buffered saline, pH 7.4 (PBS) supplemented with 0.05% Tween 20(available from Sigma Chemical Co. St. Louis, Mo.), and then blocked inPBS/T containing 5% nonfat dry milk for one hr at room temperature. Thefilters were then incubated for 3 hr with rabbit anti-B. malayitransglutaminase PBmTG20 antiserum, previously absorbed with E. coliantigens, diluted 1:500 in PBS/T. Antibody reactivity with recombinantproteins was revealed by incubation of the filters with alkalinephosphatase-conjugated goat-anti human IgG antibodies (available fromKirkegaard and Perry Laboratories (KPL), Gaithersburg, Md.) for 1 hr anddevelopment with a 5-bromo-4-chloro-3-indolyl phosphate/nitrobluetetrazolium substrate (BCIP/NBT, available from Life Technologies, Inc.,(LTI), Gaithersburg, Md.). Library clones that were reactive with thePBmTG20 antiserum were isolated and plaque-purified by the performanceof two additional cycles of immuno-screening, using the same procedureand reagents as disclosed here.

A plaque-purified clone including D. immitis nucleic acid sequencenDiTPx2₈₀₂ was converted into a double stranded recombinant molecule,herein denoted as pβgal-nDiTPx2₈₀₂, using ExAssist™ helper phage andSOLR™ E. coli according to the in vivo excision protocol described inthe Stratagene ZAP-cDNA® Synthesis Kit. Double stranded plasmid DNA wasprepared using an alkaline lysis protocol (Quantum Prep™ PlasmidMini-Prep Kit, BioRad Laboratories, Hercules, Cailf.). The plasmid DNAwas digested with EcoRI and XhoI restriction endonucleases to release asingle D. immitis nDiTPx2₈₀₂ DNA fragment of about 802 nucleotides insize.

The plasmid containing D. immitis nDiTPx2₈₀₂ was sequenced by the Sangerdideoxy chain termination method, using the PRISM™ Ready Dye TerminatorCycle Sequencing Kit with AmpliTaq® DNA Polymerase, FS (available fromthe Perkin-Elmer Corporation, Norwalk, Conn.). PCR extensions were donein the GeneAmp™ PCR System 9600 (available from Perkin-Elmer). Excessdye terminators were removed from extension products using theCentriflex™ Gel Filtration Cartridge (available from Advanced GeneticsTechnologies Corporation, Gaithersburg, Md.) following their standardprotocol. Samples were resuspended according to ABI protocols and wererun on a Perkin-Elmer ABI PRISM™ 377 Automated DNA Sequencer. Thefollowing nucleotide primers that anneal to the pBluescript® vector wereused to sequence this clone: two sense primers, T3X primer (denotedherein as SEQ ID NO:13) having the nucleic acid sequence, 5' AATTAACCCTCACTAAAGGG 3' and M13 reverse primer (denoted herein as SEQ ID NO:14)having the nucleotide sequence, 5' GGAAACAGCT ATGACCATG 3'; and twoantisense primers, T7X primer (denoted herein as SEQ ID NO:15) havingthe nucleotide sequence 5' GTAATACGAC TCACTATAGG GC 3' and M13 forwardprimer (denoted herein a SEQ ID NO:16) having the nucleotide sequence 5'GTAAAACGAC GGCCAGT 3', respectively. The resulting nucleic acid sequenceof nDiTPx2₈₀₂ is presented as SEQ ID NO:1 (the coding strand) and SEQ IDNO:3 (the complementary strand).

Translation of SEQ ID NO:1 yields a protein of about 235 amino acids,denoted herein as PHIS-PDiTPx2₂₃₅, the amino acid sequence of which ispresented in SEQ ID NO:2, assuming an initiation codon extending fromnucleotide 13 to nucleotide 15 of SEQ ID NO:1 and a stop codon extendingfrom nucleotide 718 to nucleotide 720 of SEQ ID NO:1. The coding region(not including the stop codon) encoding PHIS-PDiTPx2₂₃₅ is referred toherein as nDiTPx2₇₀₅, and has a nucleic acid sequence represented as SEQID NO:4 (the coding strand) and the SEQ ID NO:5 (the complementarystrand). The 5' end of SEQ ID NO:1, from nucleotide 1 through nucleotide6, comprises an untranslated sequence corresponding to a portion of thenematode 22-nucleotide splice leader (SL) followed by the start codonATG beginning at nucleotide position 13. SEQ ID NO:1 has a putativepolyadenylation signal, AATAAA, spanning from nucleotide 766 tonucleotide 781, followed by an 18-nucleotide poly A tail extending fromnucleotide 785 through nucleotide 802.

Computer analysis of the amino acid sequence of D. immitisPHIS-PDiTPx2₂₃₅ (i.e., SEQ ID NO:2) was performed using the PC/GENEsoftware package. The analysis revealed that PHIS-PDiTPx2₂₃₅ has apredicted molecular mass of about 26.5 kD and a predicted pI of 5.29.The protein encoded by SEQ ID NO:2 is predominantly hydrophilic aspredicted by the method of Hopp and Woods, Proc. Natl. Acad. Sci.(USA)., 78, 3824-3828. In addition SEQ ID NO:2 includes a Cys residue atposition 49. While not being bound by theory, this Cys residue is mostlikely the active site of PHIS-PDiTPx2₂₃₅.

A homology search of a non-redundant protein database was performed onSEQ ID NO:1, translated into all six reading frames, using the blastxprogram available through the BLAST™ network of the National Center forBiotechnology Information (NCBI) (National Library of Medicine, NationalInstitute of Health, Baltimore, Md.). This database includes SwissProt+PIR+SPupdate+GenPept+GPUpdate+PDB databases. The highest scoring matchof the homology search at the amino acid level was GenBank™ accessionnumber P52570, an Onchocerca volvulus adult TPx protein. SEQ ID NO:2 wasoptimally aligned with the sequence represented by GenBank™ accessionnumber P52570 using the "gap" program, available in the WisconsinPackage™. The alignment revealed that SEQ ID NO:2 had about 86% identityto the O. volvulus adult TPx protein over a region spanning from aboutamino acid 1 through about amino acid 235 of SEQ ID NO:2. SEQ ID NO:2was also aligned with the amino acid sequence of D. immitis TPx-1,disclosed as SEQ ID NO:2 in copending U.S. patent application Ser. No.08/602,010, ibid., using the "gap" program. Optimal alignment revealedthat a region of SEQ ID NO:2 of the present invention, spanning fromabout amino acid 1 through about amino acid 235, had about 27% identitywith the D. immitis TPx-1 amino acid sequence.

A BLASTn search of a non-redundant nucleotide database was performedusing SEQ ID NO:4. The nucleotide database includes GenBank™+EMBL+DDBJ+PDB. At the nucleotide level, the coding region representedin SEQ ID NO:4 was similar to that of the O. volvulus adult TPxnucleotide sequence, GenBank™ Accession No. U31052. Optimal alignment,using the "gap" program, revealed that a region of SEQ ID NO:4, spanningfrom about nucleotide 1 through about nucleotide 705, had about 86%identity with the coding region of the O. volvulus adult TPx nucleotidesequence. SEQ ID NO:4 was also aligned with the nucleotide sequence ofthe D. immitis TPx-1 coding region, disclosed as SEQ ID NO:4 incopending U.S. patent application Ser. No. 08/602,010, ibid., using the"gap" program. Optimal alignment revealed that a region of SEQ ID NO:4of the present invention, spanning from about nucleotide 1 through aboutnucleotide 705, had about 46% identity with the D. immitis TPx-1 codingregion nucleotide sequence.

Example 2

This Example discloses the production of a recombinant cell of thepresent invention.

Recombinant molecule pTrc-nDiTPx2₇₀₉, containing a D. immitis TPx-2nucleic acid molecule operatively linked to trc transcription controlsequences and to a fusion sequence encoding a T7 tag and apoly-histidine segment, was produced in the following manner. A709-nucleotide DNA fragment containing nucleotides spanning from 12through 720 of SEQ ID NO:1, denoted herein as nDiTPx2₇₀₉, wasPCR-amplified from nucleic acid molecule D. immitis nDiTPx2₈₀₂, producedas described in Example 1, using sense primer TPx-2XhoI 5' CCGAGCTCGAGAATGACAAA AGGTATTTTG TTGGGT 3' (denoted herein as SEQ ID NO:17; XhoIsite in bold) and antisense primer TPx-2KpnI 5' CCATATGGTA CCTTATTTTGGATGTGCAAC CAT 3' (denoted herein as SEQ ID NO:18, Kpn I site in bold).Recombinant molecule pTrc-nDiTPx2₇₀₉ was produced by digesting thePCR-amplified DNA fragment with XhoI and KpnI restriction endonucleases,gel purifying the resulting fragment and directionally subcloning itinto expression vector pTrcHisB™ (available from Invitrogen, San Diego,Cailf.) that had been cleaved with XhoI and KpnI and gel purified.

Recombinant molecule pTrc-nDiTPx2₇₀₉ was transformed into E. coli toform recombinant cell E. coli:pTrc-nDiTPx2₇₀₉ using standard techniques.

Example 3

This example demonstrates the production of a D. immitis TPx-2 proteinof the present invention in a prokaryotic cell.

Recombinant cell E. coli:pTrc-nDiTPx2₇₀₉, produced as described inExample 2, was cultured in shake-flasks containing an enriched bacterialgrowth medium containing 0.1 mg/ml ampicillin at about 37° C. When thecells reached an OD₆₀₀ of about 0.5, expression of D. immitispTrc-nDiTPx2₇₀₉ was induced by addition of about 0.5 mM IPTG, and thecells were cultured for about 3 hr at about 37° C. Protein productionwas monitored by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ofrecombinant cell lysates, followed by Coomassie blue staining, usingstandard techniques. Recombinant cell E. coli:pTrc-nDiTPx2₇₀₉ produced afusion protein, denoted herein as PHIS-PDiTPx2₂₃₅, that migrated with anapparent molecular weight of about 30 kD.

Immunoblot analysis of recombinant cell E. coli:pTrc-nDiTPx2₇₀₉ lysatesindicated that the about 30 kD-protein was able to bind to a T7 tagmonoclonal antibody (available from Novagen, Inc., Madison, Wis.)directed against the fusion portion of the recombinant PHIS-PDiTPx2₂₃₅fusion protein.

The PHIS-PDiTPx2₂₃₅ histidine fusion protein was separated from E. coliproteins by cobalt chelation chromatography using Talon™ Metal AffinityResin (available from CLONTECH Laboratories, Inc., Palo Alto, Cailf.) asper manufacturer's instructions, with an imidazole gradient elution.Immunoblot analyses of the E. coli:pTrc-pDiTPx2₇₀₉ lysates and thecolumn eluate indicated that the PHIS-PDiTPx2₂₃₅ 30 kD-protein isolatedusing cobalt column chromatography was able to selectively bind to a T7tag monoclonal antibody.

Example 4

This Example discloses the purification of a D. immitis TPx-2 fusionprotein of the present invention from total cell lysates, and thepreparation of rabbit antiserum that selectively binds to a D. immitisTPx-2 protein of the present invention.

TPx-2 fusion protein PHIS-PDiTPx2₂₃₅, produced as described in Example3, was isolated from E. coli proteins by cobalt chelation chromatographyas described in Example 3. Eluted fractions containing the 30-kD proteinwere pooled and dialyzed against 1X PBS to produce cobaltcolumn-purified PHIS-PDiTPx2₂₃₅. The dialyzed protein was concentratedusing a 10K molecular weight cut off Centrifugal Ultra-free®concentrator (available from Millipore Corporation, Bedford, Mass.). Thepurified protein migrated as a single 30 kD-protein band as observed byCoomassie blue staining of SDS PAGE gels as well as by immunoblotanalysis as described in Example 3.

Rabbit-anti-DiTPx-2 antiserum was produced as follows. A rabbit wasimmunized subcutaneously, first with approximately 75 μg of thePHIS-PDiTPx2₂₃₅ protein, purified as described above, emulsified withcomplete Freund's Complete Adjuvant (available from Sigma), and thenwith three subsequent booster immunizations of the same approximate doseof PHIS-PDiTPx2₂₃₅ protein emulsified in Incomplete Freund's Adjuvant(available from Sigma). The rabbit was immunized at two-week intervalsbled on alternate weeks from the immunizations. The antiserum from eachbleed were separated and stored at -70° C. until use.

Immunoglobulin G (IgG) fractions of the rabbit anti-DiTPx-2 antiserumisolated from the day 57 post-first-immunization bleed (anti-DiTPx-2IgG), and of pre-immune serum from the same rabbit were enriched for byprecipitation of the serum samples in 50% ammonium sulfate. Theprecipitated IgG fractions were each dissolved in a volume of steriledistilled H₂ O equivalent to the original serum volume. Ammonium ionswere removed from the dissolved IgG solutions by extensive dialysis in0.1 M PBS, pH 7.2.

Example 5

This Example describes the PCR amplification and subsequent isolation ofTPx-2 nucleic acid molecules from D. immitis stage-specific cDNAexpression libraries.

D. immitis TPx-2 nucleic acid molecules were PCR amplified from D.immitis life stage-specific cDNA expression libraries constructed from48-hour L3, adult male and adult female mRNAs, as follows. D. immitis48-hour L3 adult male, and adult female cDNA libraries were subjected toPCR amplification using a sense primer having SEQ ID NO:17 and anantisense primer having SEQ ID NO:18 as described in Example 2. Theprimers amplified nucleic acid molecules of the expected size (732nucleotides) from all three cDNA libraries tested, suggesting that theDirofilaria immitis TPx-2 gene is expressed in all of these life stages.

Example 6

This example describes the PCR amplification and subsequent isolation ofTPx-2 nucleic acid molecules from D. immitis cDNA libraries using thenematode 22-nucleotide splice leader.

D. immitis TPx-2 nucleic acid molecules were PCR amplified from 48-hourL3 and adult female cDNA libraries as disclosed in Example 5, using asense primer corresponding to the nematode splice leader (SL). Most, butnot all nematode messenger RNAs have the SL at their 5' ends, and thepresence of the 5' SL sequence is indicative of an apparent full lengthcDNA. See, for example Blaxter and Liu, 1996, Int. J. Parasitol. 26,1025-1033, which is incorporated herein by reference. Two primers wereused in the PCR amplification: a sense primer representing the SLsequence, having the nucleotide sequence 5' GGTTTAATTA CCCAAGTTTG AG 3'(denoted herein as SEQ ID NO:19); and an antisense primer having SEQ IDNO:18, as described in Example 2. A 736-base-pair (bp) product wasamplified from both the 48-hour L3 and adult female cDNA libraries,denoted herein as nDiTPx2(48-h L3)₇₃₆ and nDiTPx2₇₃₆, respectively.

Nucleic acid molecules nDiTPx2(48hL3)₇₃₆ and nDiTPx2₇₃₆ were gelpurified, cloned into the pCRII™ cloning vector (available fromInvitrogen) and nDiTPx2₇₃₆ was sequenced by methods disclosed inExample 1. Sequence analysis showed that nDiTPx2₇₃₆, denoted herein asSEQ ID NO:6 (the coding strand) and SEQ ID NO:7 (the complementarystrand) was identical to the corresponding region of D. immitisnDiTPx2₈₀₂, i.e., SEQ ID NO:1 and SEQ ID NO:3, described in Example 1.The fact that nucleic acid molecule nDiTPx2₇₃₆ could be amplified fromthe adult female cDNA library with the SL primer demonstrates that theoriginal messenger RNA from which nDiTPx2₇₃₆ was amplified had the 5' SLsequence, and therefore, that the coding region represented in nucleicacid molecules nDiTPx2₈₀₂ and nDiTPx2₇₃₆ are full-length. A compositenucleic acid molecule representing an apparently full-length D. immitisTPx-2 cDNA molecule is assembled by overlapping nucleic acid moleculesnDiTPx2₈₀₂ and nDiTPx2₇₃₆. This composite nucleic acid molecule isdenoted herein as nDiTPx2₈₁₈, and has nucleic acid sequence SEQ ID NO:20(the coding strand) and SEQ ID NO:21 (the complementary strand).

Example 7

This Example demonstrates the use of D. immitis nucleic acid moleculesof the present invention to obtain, by PCR amplification, a TPx-2nucleic acid molecule from a related filariid nematode, Brugia malayi.

A B. malayi TPx-2 nucleic acid molecule was PCR amplified from afirst-strand cDNA synthesis of B. malayi adult female messenger RNA,prepared by standard methods. Two primers were used for PCRamplification, a sense primer representing the SL sequence, SEQ ID NO:19as described in Example 6, and an antisense primer having SEQ ID NO:18,as described in Example 2. An about 736-bp nucleic acid molecule wasamplified from the B. malayi adult female first-strand cDNA, denotedherein as nBmTPx2₇₃₆.

Nucleic acid molecule nBmTPx2₇₃₆ was gel purified, cloned into thepCRII™ cloning vector (available from Invitrogen) and sequenced asdescribed in Example 1. The sequence is presented as SEQ ID NO:8 (thecoding strand) and SEQ ID NO:10 (the complementary strand). Translationof SEQ ID NO:8 yields an apparently full-length polypeptide of about 235amino acids, denoted PBmTPx2₂₃₅, assuming an initiation codon extendingfrom nucleotide 29 through nucleotide 31 of SEQ ID NO:8, and atermination codon extending from nucleotide 734 through nucleotide 736of SEQ ID NO:8. The resulting amino acid sequence is presented as SEQ IDNO:9. The coding region (not including the stop codon) encodingPBmTPx2₂₃₅ is referred to herein as nBmTPx2₇₀₅, and has a nucleic acidsequence represented as SEQ ID NO:11 (the coding strand) and the SEQ IDNO:12 (the complementary strand). The 5' end of SEQ ID NO:8, fromnucleotide 1 through nucleotide 22, is an untranslated sequencecorresponding to the sequence of the nematode 22 nucleotide spliceleader, SL1, followed by the start codon ATG beginning at nucleotideposition 29.

Computer analysis of the amino acid sequence of D. immitis PBmTPx2₂₃₅(i.e. SEQ ID NO:9) was performed using the PC/GENE software package. Theanalysis revealed that PBmTPx2₂₃₅ has a predicted molecular mass ofabout 26.4 kD and a predicted pI of 5.29. The protein encoded by SEQ IDNO:9 is predominantly hydrophilic as predicted by the method of Hopp andWoods, ibid., and there is one predicted N-glycosylation site at aminoacid position 117. In addition SEQ ID NO:9 includes a Cys residue atposition 49. While not being bound by theory, this Cys residue is mostlikely the active site of PBmTPx2₂₃₅.

A homology search of a non-redundant protein database was performed onSEQ ID NO:9, using the blastp program available through the BLAST™network. Some similarity was found between SEQ ID NO:9 and certain TPxproteins of eukaryotic origin. The highest scoring match of the homologysearch at amino acid level was GenBank™ accession number P52570, anOnchocerca volvulus adult TPx protein. SEQ ID NO:9 was optimally alignedwith the sequence represented by GenBank™ accession number P52570 usingthe "gap" program available in the Wisconsin Package™. The alignmentrevealed that SEQ ID NO:9 had about 81% identity to the O. volvulusadult TPx protein over a region spanning from about amino acid 1 throughabout amino acid 235 of SEQ ID NO:9. SEQ ID NO:9 was also compared tothe D. immitis TPx-2 amino acid sequence, SEQ ID NO:2 of the presentinvention. These sequences showed about 85% identity spanning from aminoacid 1 through about amino acid 235 of both sequences. SEQ ID NO:9 wasalso aligned with the amino acid sequence of D. immitis TPx-1, disclosedas SEQ ID NO:2 in copending U.S. patent application Ser. No. 08/602,010,ibid. Optimal alignment revealed that a region of SEQ ID NO:9, spanningfrom about amino acid 1 through about amino acid 235, had about 27%identity with the D. immitis TPx-1 amino acid sequence.

A BLASTn search of a non-redundant nucleotide database was performedusing SEQ ID NO:11. At the nucleotide level, the coding regionrepresented in SEQ ID NO:11 was similar to that of the O. volvulus adultTPx nucleotide sequence, GenBank™ Accession No. U31052. Optimalalignment using the "gap" program revealed that a region of SEQ IDNO:11, spanning from about nucleotide 1 through about nucleotide 705,had about 84% identity with the coding region of the O. volvulus adultTPx nucleotide sequence. SEQ ID NO:11 was also aligned with the D.immitis TPx-2 coding region represented by SEQ ID NO:4 of the presentinvention. These two sequences shared about 85% identity over a regionspanning from nucleotide 1 through nucleotide 705 of each sequence. SEQID NO:11 was also aligned with the nucleotide sequence of the D. immitisTPx-1 coding region, disclosed as SEQ ID NO:4 in copending U.S. patentapplication Ser. No. 08/602,010, ibid., using the "gap" program. Optimalalignment revealed that a region of SEQ ID NO:11, spanning from aboutnucleotide 1 through about nucleotide 705, had about 48% identity withthe D. immitis TPx-1 coding region nucleotide sequence.

Example 8

This Example describes an experiment performed in order to confirm theD. immitis genomic origin of the isolated TPx-2 cDNA nucleic acidmolecule nDiTPx2₈₀₂ and to identify genomic restriction fragmentscorresponding to nDiTPx2₈₀₂.

D. immitis genomic DNA samples were subjected to Southern blot analysisas follows. A Southern blot was prepared with gel lanes containing about10 μg of D. immitis genomic DNA restricted with EcoRI, XhoI or HindIII.The Southern Blot was hybridized under stringent hybridizationconditions with a probe consisting of nucleic acid molecule nDiTPx2₇₀₅labeled with a chemiluminescent label (ECL labeling kit, available fromAmersham, Arlington Heights, Ill.). The probe detected a single band of8.0 kilobase pairs (kb) in the genomic DNA digested with XhoI, a singleband of 7.5 kb in the genomic DNA digested with HindIII, and two bandsat 6.0 and 1.75 kb in the genomic DNA digested with EcoRI. SincenDiTPx2₇₀₅ is known to contain a single EcoRI restriction site at aboutnucleotide 184, this result suggests that the TPx-2 gene is eitherpresent as a single copy in the D. immitis genome, or is part of aclosely-spaced gene family.

Example 9

This Example describes the identification of D. immitis poly (A)⁺ RNAtranscripts corresponding to nDiTPx2₈₀₂.

A northern blot was performed as follows. D. immitis adult female andmale total RNA (8 μg each) and adult female and male poly (A)⁺ RNA (0.5μg each) were separated by electrophoresis on a 1% formaldehyde gel andtransferred to a N+ nylon membrane (available from Amersham). The RNAwas UV-cross-linked to the membrane using a Stratalinker® (availablefrom Stratagene). The blot was hybridized with a probe consisting ofnucleic acid molecule nDiTPx2₇₀₅, labeled as described in Example 8. Ineach of the four samples, the nDiTPx2₇₀₅ probe hybridized to a singleband of approximately 880 nucleotides as calculated by the MacVector™mobility program (MacVector™ Sequence Analysis Software, available fromOxford Molecular Group, PC, Oxford, UK).

Example 10

This Example demonstrates the enzyme activity of the recombinantPHIS-PDiTPx2₂₃₅ fusion protein based on an assay in which DNA cleavageby mixed function oxidase (MFO) systems can be inactivated by activeforms of TPx.

The thioredoxin peroxidase activity of recombinant proteinPHIS-PDiTPx2₂₃₅ was demonstrated as follows. In an assay similar tothose described in Lim et al, ibid., and Sauri et al, ibid., supercoiledpUC 18 plasmid (available from S tratagene) was exposed to an MFO systemconsisting of oxidative reactions involving thiol/Fe³⁺ /O²⁻, in thepresence of varying concentrations of purified PHIS-PDiTPx2₂₃₅, producedas described in Example 4. Nicking of the supercoiled plasmid DNA by theMFO system was suppressed in the presence of at least about 25-100 μg/mlof purified PHIS-PDiTPx2₂₃₅. In the absence of purified PHIS-PDiTPx2₂₃₅,at least a portion of the double stranded super-coiled plasmid DNA (formI) was nicked by the MFO system, converting it to a relaxed doublestranded circle (form II). This assay demonstrates that PHIS-PDiTPx2₂₃₅is an active thioredoxin peroxidase and thus nucleic acid moleculenDiTPx2₈₀₂ encodes a TPx protein having thioredoxin peroxidase activity.

Example 11

This Example describes the identification of native TPx-2 in various D.immitis developmental stages, by immunoblot analysis.

Rabbit anti-DiTPx-2 IgG, produced as described in Example 4, was used toidentify native D. immitis TPx-2 protein in D. immitis protein extracts,as follows. The material in crude extracts from D. immitis microfilariae(L1), 0-hour L3, 48-hour L3, 6-day L4, adult male and adult femaleworms, as well as excretory-secretory (E-S) products of adults and L4larvae were separated by running 5 μg protein per lane on a 10-well,4-20% gradient Tris-glycine SDS-PAGE gel at 200 volts for 1 hour. Theseparated proteins were transferred to a nitrocellulose membrane bystandard methods. After transfer, the membrane was blocked in 5% nonfatdry milk for 1 hr at 37° C. The membrane was incubated with rabbitanti-DiTPx-2 IgG at a dilution of 1:2500 in TBS/T. After a 1 hrincubation at room temperature, the blot was washed, and bound antibodywas detected using a alkaline phosphatase-labeled anti-rabbit IgGsecondary antibody (available from KPL) and the substrate NBT/BCIP(available from LTI). The immunoblot analysis revealed protein bandsmigrating at about 27 kD that selectively bound to therabbit-anti-DiTPx-2 IgG in adult male, adult female, all L3 and L4larval extracts, and in L4 and adult E-S products. No protein bands thatselectively bound to the rabbit-anti-DiTPx-2 IgG were seen in themicrofilarial extracts. These results suggest that native D. immitisTPx-2 is expressed in adult worms, in mosquito-derived L3, and in invitro-cultured L4 larvae, and that the protein is released into theextracellular milieu in vitro, at least by adult worms and L4 larvae.

Two dimensional immunoblot analysis of crude extracts from D. immitis0-hour L3, 6-day L4, adult male, and adult female worms, as well as E-Sproducts from L4 and adult worms, were carried out to determine theisoelectric point (pI) of D. immitis TPx-2 native protein. The firstdimension was an isoelectric focusing gel using a non-equilibriumequilibrium pH gradient containing ampholines of pI3.5-10 (availablefrom Pharmacia Biotech, Uppsala, Sweden). The second dimension was runon a 4-20% gradient Tris-glycine gel. The separated proteins weretransferred to a nitrocellulose membrane and immunoblotted usinganti-DiTPx-2 IgG antibody, as described in the preceding paragraph. Thetwo-dimensional immunoblot analysis of D. immitis extracts identifiedtwo spots of equivalent apparent molecular weight, with approximate pIsof 5.2 and 5.5, respectively.

Example 12

This Example describes the immuno-localization of native antigen encodedby nDiTPx2₈₀₂ by light microscopy.

The anatomic location of TPx-2 antigen in adult D. immitis wasdetermined by immunohistological staining, as follows. Male and femaleD. immitis worms were fixed in 4% paraformaldehyde (available fromSigma) in 0.1 M phosphate buffer, pH 7.2 overnight at 4° C. Fixed wormswere cut into 1-cm pieces, dehydrated and embedded in paraffin. Thintransverse sections of the worm of about 7 μm thickness were thenprepared using a microtome. The sections on glass slides weredeparaffinized and dehydrated using graded series of alcohol and finallyrehydrated in PBS. The slides were blocked for 1 hr in 0.7% of 30% H₂ O₂in PBS containing 10% ethanol, to inactivate endogenous peroxidases. Forimmuno-localization, the slides were blocked in PBS containing 10% fetalcalf serum (available from Sigma) and 3% bovine serum albumin (availablefrom Sigma) (PBS/FCS/BSA) for 1 hr at room temperature. They were thenflooded with 1:1000 dilution of anti-DiTPx-2 IgG (prepared as describedin Example 4) in PBS/BSA, and incubated overnight at 4° C. The slideswere then rinsed thoroughly with PBS and the antibody binding wasresolved using a peroxidase-labeled rabbit IgG secondary antibody andthe substrate 3', 3'-diaminobenzidine tetrahydrochloride (SigmaFast™tablets, available from Sigma). After color development, the slides weredehydrated in graded series of alcohol and cleared in xylene. The slideswere then covered with cover slips and observed under a NikonMicroPhot-FXA™ microscope (available from Nikon Corporation, Japan).Using anti-DiTPx-2 IgG antibody, the native antigen corresponding to D.immitis TPx-2 was found to be localized mainly in the lateral hypodermalcords in male and female worms. In addition, labeling was seen in theafibrillar muscle cells in males and in some areas of uterine walls infemales.

Example 13

This Example demonstrates the ability of a D. immitis TPx-2 protein ofthe present invention to selectively bind antibodies isolated from dogschronically infected or immune to Dirofilaria immitis infection.

Recombinant antigen PHIS-PDiTPx2₂₃₅, prepared as described in Example 4,was incubated in Immulon® 2 microtiter plates (available from DynatechLaboratories, Alexandria, Va.) at 1.0 μg/ml in 0.06 M carbonate buffer,pH 9.6, 100 μl/well, overnight at 4° C. The plates were blocked with0.01 M PBS (pH 7.4) with 0.05% Tween 20 (Sigma) and 5% fetal calf serum(PBS/T/FCS) for 1 hr at 37° C. Serum samples were obtained from beagledogs immune to heartworm infection, as well as from chronically infecteddogs. The dogs were rendered immune by chemically-abbreviatedinfections, as described in PCT Publication No. WO 94/15593, ibid. Theimmune and infected dog serum samples were diluted 1:25 in PBS/T/FCS andwere added to the first row of the ELISA plates. Two-fold dilutions werecarried out throughout the remaining rows. After 1 hr incubation at 37°C., the plates were washed with PBS/T and IgG antibody binding wasdetected with a peroxidase-conjugated anti-dog IgG antibody (availablefrom KPL). After 1 hr incubation, the plates were washed ando-phenyldiamine/H₂ O₂ substrate was added (available from Amresco®,Solon, Ohio). The enzyme reaction was stopped after 5 min at roomtemperature with 4M H₂ SO₄. Optical density (OD) was read versus a PBSblank at 490 nm with an ELISA reader, for example, a SpectraMax™ 250,available from Molecular Devices, Sunnyvale, Cailf. Both immune dogserum samples (n=4) and infected dog serum samples (n=2) had detectablelevels of IgG antibodies reactive with PHIS-PDiTPx2₂₃₅. The meanantibody levels in immune dogs, however, were significantly higher thanin the infected dogs at similar time points.

Example 14

This Example further characterizes the enzyme activity of a D. immitisTPx-2 protein of the present invention, and shows that, unlike otherTPx's, D. immitis TPx-2 does not require DTT for activity.

PHIS-PDiTPx2₂₃₅ was tested for its ability to remove H₂ O₂ in an invitro assay system, as follows. The assay was similar to that describedby Lim et al, ibid. Briefly, reaction mixtures containing varyingconcentrations of PHIS-PDiTPx2₂₃₅ in 100 mM Hepes buffer, pH 7.0(available from Sigma) in a final volume of 100 μl were incubated at 37°C. for 30 min. After incubation, H₂ O₂ at a final concentration of 2.5mM (except where indicated) was added to the reactions mixtures, whichwere incubated for an additional 30 min (except where indicated) at 37°C. The reactions were stopped by the addition of 75 μl trichloroaceticacid solution (12.5%, v/v) followed by the addition of a mixture of 30μl of 10 mM Fe(NH₄)₂ (SO₄)₂ and 20 μl of 2.5 M KSCN, which reacts withresidual H₂ O₂ to form a complex with a purple color. The removal of H₂O₂ by PHIS-PDiTPx2₂₃₅ was monitored by measuring the decrease inabsorbance at 490 nm, which is the absorbance maximum of the purplecomplex. The percent removal was calculated based on the change in A₄₉₀with added PHIS-PDiTPx2₂₃₅, relative to A₄₉₀ without addedPHIS-PDiTPx2₂₃₅ (i.e., the absorbance maximum of the complex).

PHIS-PDiTPx2₂₃₅ was able to catalyze the removal of H₂ O₂ and the effectwas concentration dependent (Table 1). Furthermore, 1.25 mg/ml ofPHIS-PDiTPx2₂₃₅ was capable of removing over 80% of 2.5 mM H₂ O₂ (Table2). Time-course studies indicated that over 60% of 2.5 mM H₂ O₂ wasremoved by PHIS -PDiTPx2₂₃₅ in less than 20 minutes (Table 3).

Other researchers have observed that a reducing agent, e.g.,dithiothreitol (DTT), is required for TPx activity; see, for example,Lim, et al., ibid. While not being bound by theory, DTT does not appearto be required for activity of the D. immitis TPx-2 protein. Although atlower concentrations it appeared that PHIS-PDiTPx2₂₃₅ was able to removeH₂ O₂ more efficiently in the presence of 10 mM DTT, at higherconcentrations of PHIS-PDiTPx2₂₃₅ (i.e., >1.25 mg/ml), DTT had no effecton the efficiency of H₂ O₂ removal (Table 4). While not being bound bytheory, the apparent enhancement of H₂ O₂ removal by lowerconcentrations of PHIS-PDiTPx2₂₃₅ in the presence of DTT may representH₂ O₂ removal by DTT itself. In control reactions without any addedPHIS-PDiTPx2₂₃₅, the maximum absorbance of the reaction complex withadded DTT was lower than that obtained without added DTT, i.e., the meanA₄₉₀ with added DTT was 3.27 while the mean A₄₉₀ without added DTT was4.2.

In inhibition studies, N-ethylmaleimide (NEM) prevented PHIS-PDiTPx2₂₃₅from removing H₂ O₂ and the inhibition was concentration dependent(Table 5). Since NEM binds to sulfhydryl group of cysteine residues,these results suggest that the sulfhydryl of cysteine in PHiS-PDiTPx2₂₃₅could function as a strong nucleophile to attack and destroy H₂ O₂.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

                  TABLE 1                                                         ______________________________________                                        Effect of PHIS-PDiTPx2.sub.235 concentration of removal of H.sub.2            O.sub.2 *                                                                          Concentration of                                                           PHIS-PDiTPx2.sub.235                                                          (μg/ml) Percent removal (Mean ± SD)                                   ______________________________________                                         0           0                                                                  250 0                                                                         500 0                                                                         750 2.97 ± 0.08                                                            1000  58.5 ± 8.84                                                          1250  83.4 ± 3.92                                                        ______________________________________                                         *H.sub.2 O.sub.2 was used at a final concentration of 2.5 mM             

                  TABLE 2                                                         ______________________________________                                        Effect of H.sub.2 O.sub.2 concentration on peroxidase                           activity of PHIS-PDiTPx2.sub.235 *                                                Concentration of H.sub.2 O.sub.2                                                            Percent removal                                             (mM) (Mean ± SD)                                                         ______________________________________                                        0.5             99.4 ± 0.01                                                  1.0 97.9 ± 0.81                                                            2.0 95.7 ± 0.30                                                            2.5 84.3 ± 0.66                                                            3.0 81.9 ± 0.87                                                            5.0 51.8 ± 2.61                                                            7.5 20.2 ± 2.80                                                            10.0  0.0 ± 0.0                                                          ______________________________________                                         *PHIS-PDiTPx2.sub.235 was used at a final concentration of 1.25 mg/ml    

                  TABLE 3                                                         ______________________________________                                        Effect of time on removal of H.sub.2 O.sub.2 .sup.†  by PHIS-PDiTPx    2.sub.235 *                                                                         Time (min)                                                                              Percent removal (mean ± SD)                                ______________________________________                                         0          0                                                                    5  6.8 ± 0.59                                                             10 43.6 ± 1.13                                                             20 64.8 ± 1.56                                                             30 82.3 ± 3.09                                                             40 87.3 ± 4.32                                                           ______________________________________                                         .sup.† Final concentration of H.sub.2 O.sub.2 used was 2.5 mM          *PHISPDiTPx2.sub.235 was used at a final concentration of 1.25 mg/ml     

                  TABLE 4                                                         ______________________________________                                        Effect of dithiothreitol (DTT) on removal of H.sub.2 O.sub.2 .sup.†     by PHIS-PDiTPx2.sub.235                                                        PHIS-PDiTPx2.sub.235     Percent removal (Mean ± SD)                     (μg/ml)      -DTT      +DTT*                                               ______________________________________                                          0             0         0                                                      250 0 18.6 ± 2.67                                                          500 1.46 ± 0.05 40.1 ± 1.97                                             750 36.1 ± 1.10 75.5 ± 3.02                                            1000 49.7 ± 6.23 76.4 ± 4.04                                            1250 92.2 ± 2.71 96.5 ± 2.32                                            1500 96.8 ± 1.80 97.8 ± 0.32                                            1750 98.4 ± 0.15 97.9 ± 0.33                                          ______________________________________                                         .sup.† Final concentration of H.sub.2 O.sub.2 used was 2.5 mM          *Final concentration of DTT used was 10 mM                               

                  TABLE 5                                                         ______________________________________                                        Effect of N-ethylmaleimide (NEM) on the peroxidase                              activity of PHIS-PDiTPx2.sub.235 *                                                Concentration of NEM                                                                        Percent protection                                          (mM) (Mean ± SD)                                                         ______________________________________                                        0               100.0 ± 0.00                                                 0.1 97.3 ± 2.40                                                            1.0 76.7 ± 1.13                                                            2.0 77.9 ± 1.87                                                            4.0 58.4 ± 1.57                                                            8.0 21.1 ± 0.33                                                            16.0 0.0 ± 0.0                                                             32.0 0.0 ± 0.0                                                           ______________________________________                                         *PHIS-PDiTPx2.sub.235 was used at a final concentration of 0.75 mg/ml.        This assay had DTT at a final concentration of 10 mM.                    

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES:  21                                         - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  802 nuc - #leotides                                              (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -     (ix) FEATURE:                                                                  (A) NAME/KEY:  CDS                                                            (B) LOCATION:  13..717                                               - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #1:                          - - TTTGAGTTGA AA ATG ACA AAA GGT ATT TTG TTG GGT - # GAT AAA                   - #  42                                                                                 Met Thr Ly - #s Gly Ile Leu Leu Gly Asp Lys                                     1  - #             5     - #             10                    - - TTT CCG GAT TTC CGA GCC GAA ACT AAT GAA GG - #C TTT ATT CCG                 - #  84                                                                    Phe Pro Asp Phe Arg Ala Glu Thr Asn Glu Gl - #y Phe Ile Pro                                    15 - #                 20                                     - - AGT TTC TAT GAT TGG ATT GGC AAA GAT AGT TG - #G GCA ATA TTA                 - # 126                                                                    Ser Phe Tyr Asp Trp Ile Gly Lys Asp Ser Tr - #p Ala Ile Leu                    25                 - # 30                 - # 35                              - - TTC TCT CAT CCA CGA GAT TTC ACT CCG GTT TG - #T ACC ACA GAA                 - # 168                                                                    Phe Ser His Pro Arg Asp Phe Thr Pro Val Cy - #s Thr Thr Glu                        40             - #     45             - #     50                          - - CTT GCT AGA CTG GTC CAA CTA GCA CCA GAA TT - #C AAG AAA CGA                 - # 210                                                                    Leu Ala Arg Leu Val Gln Leu Ala Pro Glu Ph - #e Lys Lys Arg                            55         - #         60         - #         65                      - - AAT GTG AAA CTG ATT GGT TTA AGT TGT GAC TC - #A GCA GAA TCG                 - # 252                                                                    Asn Val Lys Leu Ile Gly Leu Ser Cys Asp Se - #r Ala Glu Ser                            70         - #         75         - #         80                      - - CAT CGT AAA TGG GTT GAT GAT ATT ATG GCA GT - #A TGC AAA ATG                 - # 294                                                                    His Arg Lys Trp Val Asp Asp Ile Met Ala Va - #l Cys Lys Met                            85         - #         90                                             - - AAA TGT AAT GAT GGT GAT ACC TGC TGT TCA GG - #A AAT AAG CTA                 - # 336                                                                    Lys Cys Asn Asp Gly Asp Thr Cys Cys Ser Gl - #y Asn Lys Leu                    95                 - #100                 - #105                              - - CCG TTT CCA ATA ATA GCA GAT GAG AAT CGT TT - #T CTA GCT ACC                 - # 378                                                                    Pro Phe Pro Ile Ile Ala Asp Glu Asn Arg Ph - #e Leu Ala Thr                       110              - #   115              - #   120                          - - GAA TTA GGA ATG ATG GAT CCA GAT GAA CGT GA - #T GAA AAT GGT                 - # 420                                                                    Glu Leu Gly Met Met Asp Pro Asp Glu Arg As - #p Glu Asn Gly                           125          - #       130          - #       135                      - - AAC GCA TTA ACT GCA CGT TGT GTA TTC ATA AT - #T GGA CCT GAG                 - # 462                                                                    Asn Ala Leu Thr Ala Arg Cys Val Phe Ile Il - #e Gly Pro Glu                               140      - #           145      - #           150                  - - AAA ACG TTG AAA CTT TCT ATT TTA TAT CCT GC - #A ACA ACA GGA                 - # 504                                                                    Lys Thr Leu Lys Leu Ser Ile Leu Tyr Pro Al - #a Thr Thr Gly                                   155  - #               160                                     - - CGA AAT TTC GAT GAA ATT CTG CGC GTC GTT GA - #T TCG CTT CAA                 - # 546                                                                    Arg Asn Phe Asp Glu Ile Leu Arg Val Val As - #p Ser Leu Gln                   165                 1 - #70                 1 - #75                            - - CTT ACA GCA GTT AAA CTA GTA GCG ACA CCA GT - #C GAT TGG AAA                 - # 588                                                                    Leu Thr Ala Val Lys Leu Val Ala Thr Pro Va - #l Asp Trp Lys                       180              - #   185              - #   190                          - - GGT GGT GAT GAT TGT GTC GTG CTG CCA ACG AT - #T GAT GAT ACG                 - # 630                                                                    Gly Gly Asp Asp Cys Val Val Leu Pro Thr Il - #e Asp Asp Thr                           195          - #       200          - #       205                      - - GAG GCA AAA AAA TTG TTT GGA GAA AAG ATA AA - #T ACT ATC GAA                 - # 672                                                                    Glu Ala Lys Lys Leu Phe Gly Glu Lys Ile As - #n Thr Ile Glu                               210      - #           215      - #           220                  - - TTG CCA TCT GGA AAA CAT TAT CTT CGC ATG GT - #T GCA CAT CCA                 - # 714                                                                    Leu Pro Ser Gly Lys His Tyr Leu Arg Met Va - #l Ala His Pro                                   225  - #               230                                     - - AAA TAA AACATCATTT TGTTGCATTT TATGTTCATT TATGTTTCAT  - #                    760                                                                        Lys                                                                           235                                                                            - - TTTTCAATAA AAAATTAAAT TTGTAAAAAA AAAAAAAAAA AA    - #                      - # 802                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  235 ami - #no acids                                              (B) TYPE:  amino aci - #d                                                     (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #2:                          - - Met Thr Lys Gly Ile Leu Leu Gly Asp Lys Ph - #e Pro Asp Phe                1               5 - #                 10                                     - - Arg Ala Glu Thr Asn Glu Gly Phe Ile Pro Se - #r Phe Tyr Asp               15                 - # 20                 - # 25                              - - Trp Ile Gly Lys Asp Ser Trp Ala Ile Leu Ph - #e Ser His Pro                   30             - #     35             - #     40                          - - Arg Asp Phe Thr Pro Val Cys Thr Thr Glu Le - #u Ala Arg Leu                       45         - #         50         - #         55                      - - Val Gln Leu Ala Pro Glu Phe Lys Lys Arg As - #n Val Lys Leu                           60     - #             65     - #             70                  - - Ile Gly Leu Ser Cys Asp Ser Ala Glu Ser Hi - #s Arg Lys Trp                               75 - #                 80                                     - - Val Asp Asp Ile Met Ala Val Cys Lys Met Ly - #s Cys Asn Asp               85                 - # 90                 - # 95                              - - Gly Asp Thr Cys Cys Ser Gly Asn Lys Leu Pr - #o Phe Pro Ile                  100              - #   105              - #   110                          - - Ile Ala Asp Glu Asn Arg Phe Leu Ala Thr Gl - #u Leu Gly Met                      115          - #       120          - #       125                      - - Met Asp Pro Asp Glu Arg Asp Glu Asn Gly As - #n Ala Leu Thr                          130      - #           135      - #           140                  - - Ala Arg Cys Val Phe Ile Ile Gly Pro Glu Ly - #s Thr Leu Lys                              145  - #               150                                     - - Leu Ser Ile Leu Tyr Pro Ala Thr Thr Gly Ar - #g Asn Phe Asp               155                 - #160                 - #165                             - - Glu Ile Leu Arg Val Val Asp Ser Leu Gln Le - #u Thr Ala Val                  170              - #   175              - #   180                          - - Lys Leu Val Ala Thr Pro Val Asp Trp Lys Gl - #y Gly Asp Asp                      185          - #       190          - #       195                      - - Cys Val Val Leu Pro Thr Ile Asp Asp Thr Gl - #u Ala Lys Lys                          200      - #           205      - #           210                  - - Leu Phe Gly Glu Lys Ile Asn Thr Ile Glu Le - #u Pro Ser Gly                              215  - #               220                                     - - Lys His Tyr Leu Arg Met Val Ala His Pro Ly - #s                          225                 2 - #30                 2 - #35                            - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  802 nuc - #leotides                                              (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #3:                          - - TTTTTTTTTT TTTTTTTTAC AAATTTAATT TTTTATTGAA AAATGAAACA  - #                  50                                                                         - - TAAATGAACA TAAAATGCAA CAAAATGATG TTTTATTTTG GATGTGCAAC  - #                 100                                                                         - - CATGCGAAGA TAATGTTTTC CAGATGGCAA TTCGATAGTA TTTATCTTTT  - #                 150                                                                         - - CTCCAAACAA TTTTTTTGCC TCCGTATCAT CAATCGTTGG CAGCACGACA  - #                 200                                                                         - - CAATCATCAC CACCTTTCCA ATCGACTGGT GTCGCTACTA GTTTAACTGC  - #                 250                                                                         - - TGTAAGTTGA AGCGAATCAA CGACGCGCAG AATTTCATCG AAATTTCGTC  - #                 300                                                                         - - CTGTTGTTGC AGGATATAAA ATAGAAAGTT TCAACGTTTT CTCAGGTCCA  - #                 350                                                                         - - ATTATGAATA CACAACGTGC AGTTAATGCG TTACCATTTT CATCACGTTC  - #                 400                                                                         - - ATCTGGATCC ATCATTCCTA ATTCGGTAGC TAGAAAACGA TTCTCATCTG  - #                 450                                                                         - - CTATTATTGG AAACGGTAGC TTATTTCCTG AACAGCAGGT ATCACCATCA  - #                 500                                                                         - - TTACATTTCA TTTTGCATAC TGCCATAATA TCATCAACCC ATTTACGATG  - #                 550                                                                         - - CGATTCTGCT GAGTCACAAC TTAAACCAAT CAGTTTCACA TTTCGTTTCT  - #                 600                                                                         - - TGAATTCTGG TGCTAGTTGG ACCAGTCTAG CAAGTTCTGT GGTACAAACC  - #                 650                                                                         - - GGAGTGAAAT CTCGTGGATG AGAGAATAAT ATTGCCCAAC TATCTTTGCC  - #                 700                                                                         - - AATCCAATCA TAGAAACTCG GAATAAAGCC TTCATTAGTT TCGGCTCGGA  - #                 750                                                                         - - AATCCGGAAA TTTATCACCC AACAAAATAC CTTTTGTCAT TTTCAACTCA  - #                 800                                                                         - - AA                  - #                  - #                  - #                 802                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  705 nuc - #leotides                                              (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #4:                          - - ATGACAAAAG GTATTTTGTT GGGTGATAAA TTTCCGGATT TCCGAGCCGA  - #                  50                                                                         - - AACTAATGAA GGCTTTATTC CGAGTTTCTA TGATTGGATT GGCAAAGATA  - #                 100                                                                         - - GTTGGGCAAT ATTATTCTCT CATCCACGAG ATTTCACTCC GGTTTGTACC  - #                 150                                                                         - - ACAGAACTTG CTAGACTGGT CCAACTAGCA CCAGAATTCA AGAAACGAAA  - #                 200                                                                         - - TGTGAAACTG ATTGGTTTAA GTTGTGACTC AGCAGAATCG CATCGTAAAT  - #                 250                                                                         - - GGGTTGATGA TATTATGGCA GTATGCAAAA TGAAATGTAA TGATGGTGAT  - #                 300                                                                         - - ACCTGCTGTT CAGGAAATAA GCTACCGTTT CCAATAATAG CAGATGAGAA  - #                 350                                                                         - - TCGTTTTCTA GCTACCGAAT TAGGAATGAT GGATCCAGAT GAACGTGATG  - #                 400                                                                         - - AAAATGGTAA CGCATTAACT GCACGTTGTG TATTCATAAT TGGACCTGAG  - #                 450                                                                         - - AAAACGTTGA AACTTTCTAT TTTATATCCT GCAACAACAG GACGAAATTT  - #                 500                                                                         - - CGATGAAATT CTGCGCGTCG TTGATTCGCT TCAACTTACA GCAGTTAAAC  - #                 550                                                                         - - TAGTAGCGAC ACCAGTCGAT TGGAAAGGTG GTGATGATTG TGTCGTGCTG  - #                 600                                                                         - - CCAACGATTG ATGATACGGA GGCAAAAAAA TTGTTTGGAG AAAAGATAAA  - #                 650                                                                         - - TACTATCGAA TTGCCATCTG GAAAACATTA TCTTCGCATG GTTGCACATC  - #                 700                                                                         - - CAAAA                 - #                  - #                  - #               705                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  705 nuc - #leotides                                              (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #5:                          - - TTTTGGATGT GCAACCATGC GAAGATAATG TTTTCCAGAT GGCAATTCGA  - #                  50                                                                         - - TAGTATTTAT CTTTTCTCCA AACAATTTTT TTGCCTCCGT ATCATCAATC  - #                 100                                                                         - - GTTGGCAGCA CGACACAATC ATCACCACCT TTCCAATCGA CTGGTGTCGC  - #                 150                                                                         - - TACTAGTTTA ACTGCTGTAA GTTGAAGCGA ATCAACGACG CGCAGAATTT  - #                 200                                                                         - - CATCGAAATT TCGTCCTGTT GTTGCAGGAT ATAAAATAGA AAGTTTCAAC  - #                 250                                                                         - - GTTTTCTCAG GTCCAATTAT GAATACACAA CGTGCAGTTA ATGCGTTACC  - #                 300                                                                         - - ATTTTCATCA CGTTCATCTG GATCCATCAT TCCTAATTCG GTAGCTAGAA  - #                 350                                                                         - - AACGATTCTC ATCTGCTATT ATTGGAAACG GTAGCTTATT TCCTGAACAG  - #                 400                                                                         - - CAGGTATCAC CATCATTACA TTTCATTTTG CATACTGCCA TAATATCATC  - #                 450                                                                         - - AACCCATTTA CGATGCGATT CTGCTGAGTC ACAACTTAAA CCAATCAGTT  - #                 500                                                                         - - TCACATTTCG TTTCTTGAAT TCTGGTGCTA GTTGGACCAG TCTAGCAAGT  - #                 550                                                                         - - TCTGTGGTAC AAACCGGAGT GAAATCTCGT GGATGAGAGA ATAATATTGC  - #                 600                                                                         - - CCAACTATCT TTGCCAATCC AATCATAGAA ACTCGGAATA AAGCCTTCAT  - #                 650                                                                         - - TAGTTTCGGC TCGGAAATCC GGAAATTTAT CACCCAACAA AATACCTTTT  - #                 700                                                                         - - GTCAT                 - #                  - #                  - #               705                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  736 nuc - #leotides                                              (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -     (ix) FEATURE:                                                                  (A) NAME/KEY:  CDS                                                            (B) LOCATION:  29..733                                               - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #6:                          - - GGTTTAATTA CCCAAGTTTG AGTTGAAA ATG ACA AAA GGT ATT - #TTG                    46                                                                                          - #              Met Thr L - #ys Gly Ile Leu                                  - #                1  - #             5                      - - TTG GGT GAT AAA TTT CCG GAT TTC CGA GCC GA - #A ACT AAT GAA                 - #  88                                                                    Leu Gly Asp Lys Phe Pro Asp Phe Arg Ala Gl - #u Thr Asn Glu                                10     - #             15     - #             20                  - - GGC TTT ATT CCG AGT TTC TAT GAT TGG ATT GG - #C AAA GAT AGT                 - # 130                                                                    Gly Phe Ile Pro Ser Phe Tyr Asp Trp Ile Gl - #y Lys Asp Ser                                    25 - #                 30                                     - - TGG GCA ATA TTA TTC TCT CAT CCA CGA GAT TT - #C ACT CCG GTT                 - # 172                                                                    Trp Ala Ile Leu Phe Ser His Pro Arg Asp Ph - #e Thr Pro Val                    35                 - # 40                 - # 45                              - - TGT ACC ACA GAA CTT GCT AGA CTG GTC CAA CT - #A GCA CCA GAA                 - # 214                                                                    Cys Thr Thr Glu Leu Ala Arg Leu Val Gln Le - #u Ala Pro Glu                        50             - #     55             - #     60                          - - TTC AAG AAA CGA AAT GTG AAA CTG ATT GGT TT - #A AGT TGT GAC                 - # 256                                                                    Phe Lys Lys Arg Asn Val Lys Leu Ile Gly Le - #u Ser Cys Asp                            65         - #         70         - #         75                      - - TCA GCA GAA TCG CAT CGT AAA TGG GTT GAT GA - #T ATT ATG GCA                 - # 298                                                                    Ser Ala Glu Ser His Arg Lys Trp Val Asp As - #p Ile Met Ala                                80     - #             85     - #             90                  - - GTA TGC AAA ATG AAA TGT AAT GAT GGT GAT AC - #C TGC TGT TCA                 - # 340                                                                    Val Cys Lys Met Lys Cys Asn Asp Gly Asp Th - #r Cys Cys Ser                                    95 - #                100                                     - - GGA AAT AAG CTA CCG TTT CCA ATA ATA GCA GA - #T GAG AAT CGT                 - # 382                                                                    Gly Asn Lys Leu Pro Phe Pro Ile Ile Ala As - #p Glu Asn Arg                   105                 1 - #10                 1 - #15                            - - TTT CTA GCT ACC GAA TTA GGA ATG ATG GAT CC - #A GAT GAA CGT                 - # 424                                                                    Phe Leu Ala Thr Glu Leu Gly Met Met Asp Pr - #o Asp Glu Arg                       120              - #   125              - #   130                          - - GAT GAA AAT GGT AAC GCA TTA ACT GCA CGT TG - #T GTA TTC ATA                 - # 466                                                                    Asp Glu Asn Gly Asn Ala Leu Thr Ala Arg Cy - #s Val Phe Ile                           135          - #       140          - #       145                      - - ATT GGA CCT GAG AAA ACG TTG AAA CTT TCT AT - #T TTA TAT CCT                 - # 508                                                                    Ile Gly Pro Glu Lys Thr Leu Lys Leu Ser Il - #e Leu Tyr Pro                               150      - #           155      - #           160                  - - GCA ACA ACA GGA CGA AAT TTC GAT GAA ATT CT - #G CGC GTC GTT                 - # 550                                                                    Ala Thr Thr Gly Arg Asn Phe Asp Glu Ile Le - #u Arg Val Val                                   165  - #               170                                     - - GAT TCG CTT CAA CTT ACA GCA GTT AAA CTA GT - #A GCG ACA CCA                 - # 592                                                                    Asp Ser Leu Gln Leu Thr Ala Val Lys Leu Va - #l Ala Thr Pro                   175                 1 - #80                 1 - #85                            - - GTC GAT TGG AAA GGT GGT GAT GAT TGT GTC GT - #G CTG CCA ACG                 - # 634                                                                    Val Asp Trp Lys Gly Gly Asp Asp Cys Val Va - #l Leu Pro Thr                       185              - #   190              - #   195                          - - ATT GAT GAT ACG GAG GCA AAA AAA TTG TTT GG - #A GAA AAG ATA                 - # 676                                                                    Ile Asp Asp Thr Glu Ala Lys Lys Leu Phe Gl - #y Glu Lys Ile                           200          - #       205          - #       210                      - - AAT ACT ATC GAA TTG CCA TCT GGA AAA CAT TA - #T CTT CGC ATG                 - # 718                                                                    Asn Thr Ile Glu Leu Pro Ser Gly Lys His Ty - #r Leu Arg Met                               215      - #           220      - #           225                  - - GTT GCA CAT CCA AAA TAA         - #                  - #                      - # 736                                                                  Val Ala His Pro Lys                                                                           230                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  736 nuc - #leotides                                              (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #7:                          - - TTATTTTGGA TGTGCAACCA TGCGAAGATA ATGTTTTCCA GATGGCAATT  - #                  50                                                                         - - CGATAGTATT TATCTTTTCT CCAAACAATT TTTTTGCCTC CGTATCATCA  - #                 100                                                                         - - ATCGTTGGCA GCACGACACA ATCATCACCA CCTTTCCAAT CGACTGGTGT  - #                 150                                                                         - - CGCTACTAGT TTAACTGCTG TAAGTTGAAG CGAATCAACG ACGCGCAGAA  - #                 200                                                                         - - TTTCATCGAA ATTTCGTCCT GTTGTTGCAG GATATAAAAT AGAAAGTTTC  - #                 250                                                                         - - AACGTTTTCT CAGGTCCAAT TATGAATACA CAACGTGCAG TTAATGCGTT  - #                 300                                                                         - - ACCATTTTCA TCACGTTCAT CTGGATCCAT CATTCCTAAT TCGGTAGCTA  - #                 350                                                                         - - GAAAACGATT CTCATCTGCT ATTATTGGAA ACGGTAGCTT ATTTCCTGAA  - #                 400                                                                         - - CAGCAGGTAT CACCATCATT ACATTTCATT TTGCATACTG CCATAATATC  - #                 450                                                                         - - ATCAACCCAT TTACGATGCG ATTCTGCTGA GTCACAACTT AAACCAATCA  - #                 500                                                                         - - GTTTCACATT TCGTTTCTTG AATTCTGGTG CTAGTTGGAC CAGTCTAGCA  - #                 550                                                                         - - AGTTCTGTGG TACAAACCGG AGTGAAATCT CGTGGATGAG AGAATAATAT  - #                 600                                                                         - - TGCCCAACTA TCTTTGCCAA TCCAATCATA GAAACTCGGA ATAAAGCCTT  - #                 650                                                                         - - CATTAGTTTC GGCTCGGAAA TCCGGAAATT TATCACCCAA CAAAATACCT  - #                 700                                                                         - - TTTGTCATTT TCAACTCAAA CTTGGGTAAT TAAACC      - #                  -     #      736                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  736 nuc - #leotides                                              (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -     (ix) FEATURE:                                                                  (A) NAME/KEY:  CDS                                                            (B) LOCATION:  29..733                                               - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #8:                          - - GGTTTAATTA CCCAAGTTTG AGTTAACA ATG TCA AAA GGA ATC - #CTA                    46                                                                                          - #             Met Ser Lys - #Gly Ile Leu                                    - #               1   - #            5                       - - CTT GGT GAT AAA TTT CCG GAT TTC CAA GCC GA - #G ACC AGT GAA                 - #  88                                                                    Leu Gly Asp Lys Phe Pro Asp Phe Gln Ala Gl - #u Thr Ser Glu                                10     - #             15     - #             20                  - - AGC TTC ATT TCG AGT TTC CAT GAC TGG ATT GG - #T AAA GAT AGT                 - # 130                                                                    Ser Phe Ile Ser Ser Phe His Asp Trp Ile Gl - #y Lys Asp Ser                                    25 - #                 30                                     - - TGG GCA ATA TTG TTT TCT CAT CCA CGA GAT TT - #C ACT CCA GTT                 - # 172                                                                    Trp Ala Ile Leu Phe Ser His Pro Arg Asp Ph - #e Thr Pro Val                    35                 - # 40                 - # 45                              - - TGC ACC ACG GAG CTT GCT AGG CTA GTT CAA CT - #A GAG CCG GAA                 - # 214                                                                    Cys Thr Thr Glu Leu Ala Arg Leu Val Gln Le - #u Glu Pro Glu                        50             - #     55             - #     60                          - - TTC AAG AAA CGG AAT GTA AAA CTG ATT GGT TT - #A AGT TGT GAT                 - # 256                                                                    Phe Lys Lys Arg Asn Val Lys Leu Ile Gly Le - #u Ser Cys Asp                            65         - #         70         - #         75                      - - TCG GTA CAG TCG CAC CGT AAA TGG GCT GAT GA - #T ATC ATC GAA                 - # 298                                                                    Ser Val Gln Ser His Arg Lys Trp Ala Asp As - #p Ile Ile Glu                                80     - #             85     - #             90                  - - CTG TGC AGA ATG AAG TCT GGG GAT AGT AAT AC - #C TGC TGT TCA                 - # 340                                                                    Leu Cys Arg Met Lys Ser Gly Asp Ser Asn Th - #r Cys Cys Ser                                    95 - #                100                                     - - GGG AAT AAA CTG CCG TTT CCG ATA ATA GCG GA - #T GAT AAT CGT                 - # 382                                                                    Gly Asn Lys Leu Pro Phe Pro Ile Ile Ala As - #p Asp Asn Arg                   105                 1 - #10                 1 - #15                            - - TCT CTA GCC AGT AAA CTG GGA ATG ATG GAT CC - #G GAT GAG TGT                 - # 424                                                                    Ser Leu Ala Ser Lys Leu Gly Met Met Asp Pr - #o Asp Glu Cys                       120              - #   125              - #   130                          - - GAT GAA AAG GGC GCT GCG CTA ACA GCA CGT TG - #T TTG TTC ATA                 - # 466                                                                    Asp Glu Lys Gly Ala Ala Leu Thr Ala Arg Cy - #s Leu Phe Ile                           135          - #       140          - #       145                      - - ATT GGG CCT GAG AAA ACG TTG AAA CTT TCT AT - #C CTA TAT CCT                 - # 508                                                                    Ile Gly Pro Glu Lys Thr Leu Lys Leu Ser Il - #e Leu Tyr Pro                               150      - #           155      - #           160                  - - GCA ACA ACG GGA CGA AAT TTC GAT GAA ATA TT - #G CGC GTT GTT                 - # 550                                                                    Ala Thr Thr Gly Arg Asn Phe Asp Glu Ile Le - #u Arg Val Val                                   165  - #               170                                     - - GAT TCG CTT CAG CTT ACG GCA ACT AAA TTA GT - #A GCG ACA CCA                 - # 592                                                                    Asp Ser Leu Gln Leu Thr Ala Thr Lys Leu Va - #l Ala Thr Pro                   175                 1 - #80                 1 - #85                            - - GTC GAT TGG CAG AAT GGT GAT GAT TGT GTC GT - #G GTG CCA ACG                 - # 634                                                                    Val Asp Trp Gln Asn Gly Asp Asp Cys Val Va - #l Val Pro Thr                       190              - #   195              - #   200                          - - ATT AAT GAC AAT GAA GCA AAA AAA TTG TTT GG - #T GAA AAG ATA                 - # 676                                                                    Ile Asn Asp Asn Glu Ala Lys Lys Leu Phe Gl - #y Glu Lys Ile                           205          - #       210          - #       215                      - - AAT ACT GTT GAG CTG CCA TCT GGA AAA CGT TA - #T CTT CGC ATG                 - # 718                                                                    Asn Thr Val Glu Leu Pro Ser Gly Lys Arg Ty - #r Leu Arg Met                               220      - #           225      - #           230                  - - GTT GCA CAT CCA AAA TAA         - #                  - #                      - # 736                                                                  Val Ala His Pro Lys                                                                           235                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  235 ami - #no acids                                              (B) TYPE:  amino aci - #d                                                     (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  protein                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #9:                          - - Met Ser Lys Gly Ile Leu Leu Gly Asp Lys Ph - #e Pro Asp Phe                1               5 - #                 10                                     - - Gln Ala Glu Thr Ser Glu Ser Phe Ile Ser Se - #r Phe His Asp               15                 - # 20                 - # 25                              - - Trp Ile Gly Lys Asp Ser Trp Ala Ile Leu Ph - #e Ser His Pro                   30             - #     35             - #     40                          - - Arg Asp Phe Thr Pro Val Cys Thr Thr Glu Le - #u Ala Arg Leu                       45         - #         50         - #         55                      - - Val Gln Leu Glu Pro Glu Phe Lys Lys Arg As - #n Val Lys Leu                           60     - #             65     - #             70                  - - Ile Gly Leu Ser Cys Asp Ser Val Gln Ser Hi - #s Arg Lys Trp                               75 - #                 80                                     - - Ala Asp Asp Ile Ile Glu Leu Cys Arg Met Ly - #s Ser Gly Asp               85                 - # 90                 - # 95                              - - Ser Asn Thr Cys Cys Ser Gly Asn Lys Leu Pr - #o Phe Pro Ile                  100              - #   105              - #   110                          - - Ile Ala Asp Asp Asn Arg Ser Leu Ala Ser Ly - #s Leu Gly Met                      115          - #       120          - #       125                      - - Met Asp Pro Asp Glu Cys Asp Glu Lys Gly Al - #a Ala Leu Thr                          130      - #           135      - #           140                  - - Ala Arg Cys Leu Phe Ile Ile Gly Pro Glu Ly - #s Thr Leu Lys                              145  - #               150                                     - - Leu Ser Ile Leu Tyr Pro Ala Thr Thr Gly Ar - #g Asn Phe Asp              155                 1 - #60                 1 - #65                            - - Glu Ile Leu Arg Val Val Asp Ser Leu Gln Le - #u Thr Ala Thr                  170              - #   175              - #   180                          - - Lys Leu Val Ala Thr Pro Val Asp Trp Gln As - #n Gly Asp Asp                      185          - #       190          - #       195                      - - Cys Val Val Val Pro Thr Ile Asn Asp Asn Gl - #u Ala Lys Lys                          200      - #           205      - #           210                  - - Leu Phe Gly Glu Lys Ile Asn Thr Val Glu Le - #u Pro Ser Gly                              215  - #               220                                     - - Lys Arg Tyr Leu Arg Met Val Ala His Pro Ly - #s                          225                 2 - #30                 2 - #35                            - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  736 nuc - #leotides                                              (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #10:                         - - TTATTTTGGA TGTGCAACCA TGCGAAGATA ACGTTTTCCA GATGGCAGCT  - #                  50                                                                         - - CAACAGTATT TATCTTTTCA CCAAACAATT TTTTTGCTTC ATTGTCATTA  - #                 100                                                                         - - ATCGTTGGCA CCACGACACA ATCATCACCA TTCTGCCAAT CGACTGGTGT  - #                 150                                                                         - - CGCTACTAAT TTAGTTGCCG TAAGCTGAAG CGAATCAACA ACGCGCAATA  - #                 200                                                                         - - TTTCATCGAA ATTTCGTCCC GTTGTTGCAG GATATAGGAT AGAAAGTTTC  - #                 250                                                                         - - AACGTTTTCT CAGGCCCAAT TATGAACAAA CAACGTGCTG TTAGCGCAGC  - #                 300                                                                         - - GCCCTTTTCA TCACACTCAT CCGGATCCAT CATTCCCAGT TTACTGGCTA  - #                 350                                                                         - - GAGAACGATT ATCATCCGCT ATTATCGGAA ACGGCAGTTT ATTCCCTGAA  - #                 400                                                                         - - CAGCAGGTAT TACTATCCCC AGACTTCATT CTGCACAGTT CGATGATATC  - #                 450                                                                         - - ATCAGCCCAT TTACGGTGCG ACTGTACCGA ATCACAACTT AAACCAATCA  - #                 500                                                                         - - GTTTTACATT CCGTTTCTTG AATTCCGGCT CTAGTTGAAC TAGCCTAGCA  - #                 550                                                                         - - AGCTCCGTGG TGCAAACTGG AGTGAAATCT CGTGGATGAG AAAACAATAT  - #                 600                                                                         - - TGCCCAACTA TCTTTACCAA TCCAGTCATG GAAACTCGAA ATGAAGCTTT  - #                 650                                                                         - - CACTGGTCTC GGCTTGGAAA TCCGGAAATT TATCACCAAG TAGGATTCCT  - #                 700                                                                         - - TTTGACATTG TTAACTCAAA CTTGGGTAAT TAAACC      - #                  -     #      736                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  705 nuc - #leotides                                              (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #11:                         - - ATGTCAAAAG GAATCCTACT TGGTGATAAA TTTCCGGATT TCCAAGCCGA  - #                  50                                                                         - - GACCAGTGAA AGCTTCATTT CGAGTTTCCA TGACTGGATT GGTAAAGATA  - #                 100                                                                         - - GTTGGGCAAT ATTGTTTTCT CATCCACGAG ATTTCACTCC AGTTTGCACC  - #                 150                                                                         - - ACGGAGCTTG CTAGGCTAGT TCAACTAGAG CCGGAATTCA AGAAACGGAA  - #                 200                                                                         - - TGTAAAACTG ATTGGTTTAA GTTGTGATTC GGTACAGTCG CACCGTAAAT  - #                 250                                                                         - - GGGCTGATGA TATCATCGAA CTGTGCAGAA TGAAGTCTGG GGATAGTAAT  - #                 300                                                                         - - ACCTGCTGTT CAGGGAATAA ACTGCCGTTT CCGATAATAG CGGATGATAA  - #                 350                                                                         - - TCGTTCTCTA GCCAGTAAAC TGGGAATGAT GGATCCGGAT GAGTGTGATG  - #                 400                                                                         - - AAAAGGGCGC TGCGCTAACA GCACGTTGTT TGTTCATAAT TGGGCCTGAG  - #                 450                                                                         - - AAAACGTTGA AACTTTCTAT CCTATATCCT GCAACAACGG GACGAAATTT  - #                 500                                                                         - - CGATGAAATA TTGCGCGTTG TTGATTCGCT TCAGCTTACG GCAACTAAAT  - #                 550                                                                         - - TAGTAGCGAC ACCAGTCGAT TGGCAGAATG GTGATGATTG TGTCGTGGTG  - #                 600                                                                         - - CCAACGATTA ATGACAATGA AGCAAAAAAA TTGTTTGGTG AAAAGATAAA  - #                 650                                                                         - - TACTGTTGAG CTGCCATCTG GAAAACGTTA TCTTCGCATG GTTGCACATC  - #                 700                                                                         - - CAAAA                 - #                  - #                  - #               705                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  705 nuc - #leotides                                              (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #12:                         - - TTTTGGATGT GCAACCATGC GAAGATAACG TTTTCCAGAT GGCAGCTCAA  - #                  50                                                                         - - CAGTATTTAT CTTTTCACCA AACAATTTTT TTGCTTCATT GTCATTAATC  - #                 100                                                                         - - GTTGGCACCA CGACACAATC ATCACCATTC TGCCAATCGA CTGGTGTCGC  - #                 150                                                                         - - TACTAATTTA GTTGCCGTAA GCTGAAGCGA ATCAACAACG CGCAATATTT  - #                 200                                                                         - - CATCGAAATT TCGTCCCGTT GTTGCAGGAT ATAGGATAGA AAGTTTCAAC  - #                 250                                                                         - - GTTTTCTCAG GCCCAATTAT GAACAAACAA CGTGCTGTTA GCGCAGCGCC  - #                 300                                                                         - - CTTTTCATCA CACTCATCCG GATCCATCAT TCCCAGTTTA CTGGCTAGAG  - #                 350                                                                         - - AACGATTATC ATCCGCTATT ATCGGAAACG GCAGTTTATT CCCTGAACAG  - #                 400                                                                         - - CAGGTATTAC TATCCCCAGA CTTCATTCTG CACAGTTCGA TGATATCATC  - #                 450                                                                         - - AGCCCATTTA CGGTGCGACT GTACCGAATC ACAACTTAAA CCAATCAGTT  - #                 500                                                                         - - TTACATTCCG TTTCTTGAAT TCCGGCTCTA GTTGAACTAG CCTAGCAAGC  - #                 550                                                                         - - TCCGTGGTGC AAACTGGAGT GAAATCTCGT GGATGAGAAA ACAATATTGC  - #                 600                                                                         - - CCAACTATCT TTACCAATCC AGTCATGGAA ACTCGAAATG AAGCTTTCAC  - #                 650                                                                         - - TGGTCTCGGC TTGGAAATCC GGAAATTTAT CACCAAGTAG GATTCCTTTT  - #                 700                                                                         - - GACAT                 - #                  - #                  - #               705                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  20 nucl - #eotides                                               (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  primer                                           - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #13:                         - - AATTAACCCT CACTAAAGGG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  19 nucl - #eotides                                               (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  primer                                           - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #14:                         - - GGAAACAGCT ATGACCATG             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  22 nucl - #eotides                                               (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  primer                                           - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #15:                         - - GTAATACGAC TCACTATAGG GC           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  17 nucl - #eotides                                               (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  primer                                           - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #16:                         - - GTAAAACGAC GGCCAGT             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  36 nucl - #eotides                                               (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  primer                                           - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #17:                         - - CCGAGCTCGA GAATGACAAA AGGTATTTTG TTGGGT      - #                  -     #       36                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  33 nucl - #eotides                                               (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  primer                                           - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #18:                         - - CCATATGGTA CCTTATTTTG GATGTGCAAC CAT       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  22 nucl - #eotides                                               (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  primer                                           - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #19:                         - - GGTTTAATTA CCCAAGTTTG AG           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  818 nuc - #leotides                                              (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #20:                         - - GGTTTAATTA CCCAAGTTTG AGTTGAAAAT GACAAAAGGT ATTTTGTTGG  - #                  50                                                                         - - GTGATAAATT TCCGGATTTC CGAGCCGAAA CTAATGAAGG CTTTATTCCG  - #                 100                                                                         - - AGTTTCTATG ATTGGATTGG CAAAGATAGT TGGGCAATAT TATTCTCTCA  - #                 150                                                                         - - TCCACGAGAT TTCACTCCGG TTTGTACCAC AGAACTTGCT AGACTGGTCC  - #                 200                                                                         - - AACTAGCACC AGAATTCAAG AAACGAAATG TGAAACTGAT TGGTTTAAGT  - #                 250                                                                         - - TGTGACTCAG CAGAATCGCA TCGTAAATGG GTTGATGATA TTATGGCAGT  - #                 300                                                                         - - ATGCAAAATG AAATGTAATG ATGGTGATAC CTGCTGTTCA GGAAATAAGC  - #                 350                                                                         - - TACCGTTTCC AATAATAGCA GATGAGAATC GTTTTCTAGC TACCGAATTA  - #                 400                                                                         - - GGAATGATGG ATCCAGATGA ACGTGATGAA AATGGTAACG CATTAACTGC  - #                 450                                                                         - - ACGTTGTGTA TTCATAATTG GACCTGAGAA AACGTTGAAA CTTTCTATTT  - #                 500                                                                         - - TATATCCTGC AACAACAGGA CGAAATTTCG ATGAAATTCT GCGCGTCGTT  - #                 550                                                                         - - GATTCGCTTC AACTTACAGC AGTTAAACTA GTAGCGACAC CAGTCGATTG  - #                 600                                                                         - - GAAAGGTGGT GATGATTGTG TCGTGCTGCC AACGATTGAT GATACGGAGG  - #                 650                                                                         - - CAAAAAAATT GTTTGGAGAA AAGATAAATA CTATCGAATT GCCATCTGGA  - #                 700                                                                         - - AAACATTATC TTCGCATGGT TGCACATCCA AAATAAAACA TCATTTTGTT  - #                 750                                                                         - - GCATTTTATG TTCATTTATG TTTCATTTTT CAATAAAAAA TTAAATTTGT  - #                 800                                                                         - - AAAAAAAAAA AAAAAAAA             - #                  - #                      - # 818                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  818 nuc - #leotides                                              (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #21:                         - - TTTTTTTTTT TTTTTTTTAC AAATTTAATT TTTTATTGAA AAATGAAACA  - #                  50                                                                         - - TAAATGAACA TAAAATGCAA CAAAATGATG TTTTATTTTG GATGTGCAAC  - #                 100                                                                         - - CATGCGAAGA TAATGTTTTC CAGATGGCAA TTCGATAGTA TTTATCTTTT  - #                 150                                                                         - - CTCCAAACAA TTTTTTTGCC TCCGTATCAT CAATCGTTGG CAGCACGACA  - #                 200                                                                         - - CAATCATCAC CACCTTTCCA ATCGACTGGT GTCGCTACTA GTTTAACTGC  - #                 250                                                                         - - TGTAAGTTGA AGCGAATCAA CGACGCGCAG AATTTCATCG AAATTTCGTC  - #                 300                                                                         - - CTGTTGTTGC AGGATATAAA ATAGAAAGTT TCAACGTTTT CTCAGGTCCA  - #                 350                                                                         - - ATTATGAATA CACAACGTGC AGTTAATGCG TTACCATTTT CATCACGTTC  - #                 400                                                                         - - ATCTGGATCC ATCATTCCTA ATTCGGTAGC TAGAAAACGA TTCTCATCTG  - #                 450                                                                         - - CTATTATTGG AAACGGTAGC TTATTTCCTG AACAGCAGGT ATCACCATCA  - #                 500                                                                         - - TTACATTTCA TTTTGCATAC TGCCATAATA TCATCAACCC ATTTACGATG  - #                 550                                                                         - - CGATTCTGCT GAGTCACAAC TTAAACCAAT CAGTTTCACA TTTCGTTTCT  - #                 600                                                                         - - TGAATTCTGG TGCTAGTTGG ACCAGTCTAG CAAGTTCTGT GGTACAAACC  - #                 650                                                                         - - GGAGTGAAAT CTCGTGGATG AGAGAATAAT ATTGCCCAAC TATCTTTGCC  - #                 700                                                                         - - AATCCAATCA TAGAAACTCG GAATAAAGCC TTCATTAGTT TCGGCTCGGA  - #                 750                                                                         - - AATCCGGAAA TTTATCACCC AACAAAATAC CTTTTGTCAT TTTCAACTCA  - #                 800                                                                         - - AACTTGGGTA ATTAAACC             - #                  - #                      - # 818                                                                 __________________________________________________________________________

What is claimed is:
 1. An isolated TPx-2 nucleic acid molecule selectedfrom the group consisting of; (i) a Dirofilaria TPx-2 cDNA molecule:(ii) a Dirofilaria TPx-2 mRNA molecule; (iii) a Brugia TPx-2 cDNAmolecule; and (iv) a Brugia TPx-2 mRNA molecule, wherein said cDNA ormRNA molecule of (i), (ii), (iii), and (iv) encodes a protein havingthioredoxin peroxidase activity, said protein comprising an amino acidsequence that is at least 90% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:2 and SEQ ID NO:9. 2.The TPx-2 cDNA or mRNA molecule of claim 1, wherein said cDNA or mRNAmolecule is selected from the group consisting of a cDNA or mRNA thatencodes a protein having thioredoxin peroxidase activity, said proteincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:2 and SEQ ID NO:9.
 3. The TPx-2 cDNA molecule of claim 1,where said cDNA is selected from the group consisting of nDiTPx2₈₁₈,nDiTPx2₈₀₂, nDiTPx2₇₀₉, nDiTPx2,₇₀₅, nDiTPx2₇₃₆, nBmTP2₇₃₆, andnBmTPx2₇₀₅.
 4. The TPx- 2 cDNA or mRNA molecule of claim 1, wherein saidcDNA or mRNA molecule comprises a nucleic acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:20 and SEQ ID NO:21, where in said mRNAmolecule, the deoxynucleotide T of SEQ ID NO:1 SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, D NO:7. SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:20 and SEQ ID NO:21 isreplaced by the ribonucleotide U.
 5. A recombinant molecule comprising aTPx-2 cDNA or mRNA molecule as set forth in claim 1 operatively linkedto a heterologous transcription control sequence.
 6. A recombinant viruscomprising a TPx-2 cDNA or mRNA molecule as set forth in claim
 1. 7. Arecombinant cell comprising a TPx-2 cDNA or mRNA molecule as set forthin claim 1, wherein said cell is not a Dirofilaria or a Brugia cell. 8.An isolated composition comprising an isolated TPx-2 nucleic acidmolecule selected from the group consisting of; (i) a Dirofilaria TPx-2cDNA molecule; (ii) a Dirofilaria TPx-2 mRNA molecule; (iii) a BrugiaTPx-2 cDNA molecule; and (iv) a Brugia TPx-2 mRNA molecule, wherein saidcDNA or mRNA molecule of (i), (ii), (iii), and (iv) encodes a proteinhaving thioredoxin peroxidase activity, said protein comprising an aminoacid sequence that is at least 90% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:2 and SEQ ID NO:9. 9.The composition of claim 8, wherein said composition further comprises acomponent selected from the group consisting of an excipient, anadjuvant, and a carrier.
 10. A method to elicit an immune response, saidmethod comprising administering to an animal an isolated compositioncomprising an isolated TPx-2 nucleic acid molecule selected from thegroup consisting of; (i) a Dirofilaria TPx-2 cDNA molecule; (ii) aDirofilaria TPx-2 mRNA molecule; (iii) a Brugia TPx-2 cDNA molecule; and(iv) a Brugia TPx-2 mRNA molecule, wherein said cDNA or mRNA molecule of(i), (ii), (iii), and (iv) encodes a protein having thioredoxinperoxidase activity, said protein comprising an amino acid sequence thatis at least 90% identical to an amino acid sequence selected from thegroup consisting of SEQ ID NO:2 and SEQ ID NO:9.
 11. The method of claim10, wherein said composition further comprises a component selected fromthe group consisting of an excipient, an adjuvant, a carrier, and amixture thereof.
 12. A method to produce a TPx-2 protein, said methodcomprising culturing in an effective medium a cell capable of expressingsaid protein, said protein being encoded by an isolated TPx-2 nucleicacid molecule selected from the group consisting of a Dirofilaria TPx-2cDNA molecule; (ii) a Dirofilaria TPx-2 mRNA molecule; (iii a BrugiaTPx-2 cDNA molecule; and (iv) a Brugia TPx-2 mRNA molecule, wherein saidcDNA or mRNA molecule of (i, (ii), (iii), and (iv) encodes a proteinhaving thioredoxin peroxidase activity, said protein comprising an aminoacid sequence that is at least 90% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:2 and SEQ ID NO:9.